Objective Response after Immune Checkpoint Inhibitors in a Chemotherapy-Refractory pMMR/MSS Metastatic Rectal Cancer Patient Primed with Experimental AlloStim® Immunotherapy.

Objective Response after Immune Checkpoint Inhibitors in a Chemotherapy-Refractory pMMR/MSS Metastatic Rectal Cancer Patient Primed with Experimental AlloStim® Immunotherapy. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Objective Response after Immune Checkpoint Inhibitors in a Chemotherapy-Refractory pMMR/MSS Metastatic Rectal Cancer Patient Primed with Experimental AlloStim® Immunotherapy. Azriel Hirschfeld, Daniel Gurell, Michael Har-Noy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4166800/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background : Immune Checkpoint Inhibitor (ICI) immunotherapy is most effective in immune effector cell infiltrated ‘hot’ tumor lesions, such as occurs in deficient mismatch repair, microsatellite instability high (dMMR/MSI-H) colorectal cancer (CRC). However, most all metastatic CRC tumors are mismatch repair proficient/microsatellite stable (pMMR/MSS) ‘cold’ lesions, without significant immune cell infiltration, and are unresponsive to ICI. AlloStim®, is an experimental, allogeneic immunomodulatory cell therapy designed to convert ‘cold’ metastatic tumor lesions to ‘hot’ inflamed lesions. After AlloStim® immunotherapy, this cold to hot inflammatory mechanism can make it difficult to distinguish between pseudoprogression and actual progression on restaging CT scans, as inflamed metastatic lesions can appear larger and occult disease can appear as new small lesions. Methods : To explore whether radiological progression after AlloStim® immunotherapy is due to immune-flare or disease progression, we administered a short course of a combination ICI therapy to a pMMR/MSS chemotherapy-refractory metastatic colorectal cancer patient enrolled in the StimVax Phase IIb clinical study that presented with radiological progression after AlloStim® immunotherapy. Our rationale was that an accelerated response to ICI should occur if the lesions were inflamed, while if the enlarged lesions were due to disease progression there would not be a response. Results : Here we report a rapid, significant reduction in tumor burden in response to ICI administration in an AlloStim ® primed pMMR/MSS mCRC patient with retroperitoneal and lung metastases. Conclusion : This rare objective response to ICIs in a pMMR/MSS mCRC patient supports further evaluation of the combination of AlloStim ® with ICI immunotherapy in MSS mCRC and other cold or ICI refractory tumors. Trial registration: National Library of Medicine (NLM) at the National Institutes of Health (NIH). Registered 22 June 2020, https://clinicaltrials.gov/study/NCT04444622 immunotherapy colorectal cancer microsatellite stable checkpoint inhibitor microsatellite stable mis-match repair proficient Figures Figure 1 Figure 2 Figure 3 BACKGROUND Immune checkpoint inhibitor (ICI)-based regimens have not yet shown meaningful positive outcomes in proficient DNA mismatch repair/microsatellite stable (pMMR/MSS) metastatic colorectal cancers (mCRC). Here we report a rare objective response in a pMMR/MSS heavily pre-treated metastatic colorectal cancer (mCRC) patient subsequent to a short course of an immune checkpoint inhibitor (ICI) combination after first being primed with an experimental immunomodulatory cell therapy drug, AlloStim®, designed to convert immunologically ‘cold’ tumors to ‘hot’ tumors, and a short, low dose course of regorafenib. The FDA has approved ICI drugs targeting CTLA-4, PD-1, PD-L1 and LAG-3 checkpoint molecules for a variety of solid tumor indications, including melanoma, renal, bladder, lung, gastric, gastroesophageal junction, hepatocellular carcinoma and head and neck cancers. However, ICIs have demonstrated only limited efficacy in mCRC. An anti-CTLA4 ICI, pembrolizumab, was approved in first line mCRC [ 1 ] and also approved in combination with the anti-PD-1 ICI, nivolumab, for a small subset of mCRC patients that have deficient DNA mismatch repair/microsatellite instability-high (dMMR/MSI-H) status [ 2 – 8 ]. The dMMR/MSI-H subset constitutes only ~ 5% of mCRC patients [ 9 ], while the remaining ~ 95% that present with pMMR/MSS status do not respond to ICI immunotherapy [ 6 , 8 , 10 – 13 ]. Most dMMR/MSI-H status tumors are considered immunologically ‘hot’ tumors, while pMMR/MSS status tumors are considered to be immunologically ‘cold’ [ 14 ]. Hot tumors are characterized by an inflamed phenotype, including a high level of infiltrating T-cells and NK cells, an interferon-γ (IFN-γ) signature and upregulated PD-L1 expression, while cold tumors have an absence of tumor-infiltrating lymphocytes [ 15 ]. While dMMR/MSI-H mCRC patients are more responsive to ICI therapy, approximately 50% are refractory [ 16 – 18 ]. Resistance to ICI responsiveness, regardless of MMR/MSI status, is correlated with tumor mutational burden (TMB) [ 19 ]. Since somatic mutations can encode immunogenic neoantigens, high TMB is believed to be more likely to prime for infiltrating tumor-specific effector immune cells. Consistent with this, the dMMR/MSI-H patients that present with lower TMB values have been shown to be the non-responders, whereas patients with the highest TMB values tend to obtain benefit from ICI [ 16 ], particularly with anti-CTLA-4/PD-1 combination ICI immunotherapy[ 20 ]. Present strategies for increasing the effectiveness of ICI immunotherapy in pMMR/MSS mCRC cold tumors include evaluating combinations with other therapeutic methods, such as chemotherapy, targeted therapy, and radiotherapy [ 21 ]. In addition, strategies to convert immunologically cold tumors to hot tumors are thought to be a fruitful line of investigation for increasing ICI efficacy in ICI refractory tumors [ 22 , 23 ] . AlloStim® is an experimental cellular immunotherapy drug designed to convert immunologically cold tumors to hot tumors by mirroring the graft vs. tumor (GVT) mechanism of allogeneic stem cell transplant procedures to create a host vs tumor (HVT) effect without graft vs. host disease (GVHD) toxicity [ 24 ]. AlloStim® is currently being evaluated as a monotherapy in the STIMVAX Phase IIB open label clinical trial in third-line chemotherapy-refractory pMMR/MSS mCRC (NCT04444622). The primary end-point in this study is overall survival (OS) and an exploratory end-point is objective tumor response by RECIST 1.1. AlloStim® is a living, allogeneic (“off-the-shelf”), non-genetically manipulated, activated Th1-like immune cell therapy derived from CD4 + T-cell precursors isolated from the blood of healthy donors. The STIMVAX protocol provides for three monthly cycles of weekly AlloStim® administration (intradermal and intravenous) designed to modulate the immune character of cancer patients, including increase in circulating memory Th1/Th2 ratio [ 25 ], activation of circulating memory T cells and NK cells, causing their extravasation to tumor sites [ 24 ]. The anti-tumor effects of this immunomodulation are correlated with the establishment of an IFN-γ dominated microenvironment [ 26 ]. The intravenous infusion and rejection of AlloStim® in primed allo-immune patients releases cytokines which serve to non-specifically activate memory cells and NK cells. Activated memory cells will extravasate and localize at sites of inflammation, including tumor lesions. This systemic tumor infiltration mechanism is believed to convert immunologically cold tumors to hot tumors. The modulation of the tumor microenvironment (TME) can also counter-regulate tumor-mediated immune suppression [ 27 ]. Infiltrating memory T-cells and NK cells can produce intratumoral type I cytokines, including IL-12 and IFN-γ. These cytokines can act to: upregulate MHC-I on tumor cells making them susceptible to CD8 + T-cell recognition; support immunological tumor cell death releasing neoantigens and danger signals into the TME [ 28 , 29 ]; cause maturation of dendritic cells that engulf and process chaperoned tumor neoantigens to DC1 (IL-12 + CD80/86, MHC I and MHC II positive); and convert M2 macrophages to M1 [ 30 ]. Tumor lysis that causes the release of internal chaperoned neoantigens into an inflammatory TME creates the conditions to support an in-situ vaccination mechanism [ 31 ]. In-situ vaccination involves immature dendritic cells processing the released chaperoned neoantigens to display on MHC I and MHC II, to mature to IL-12 + DC1 and migrate to the draining lymph nodes, resulting in a patient-specific anti-tumor adaptive immune response [ 32 ]. In previous clinical studies, the inflammatory mechanism of AlloStim® almost always caused post-treatment CT scan images to be read as progressive disease (PD), with systemic increases in size of existing tumor lesions and often the appearance of new small lesions (especially in lungs). However, this PD determination did not always correlate with the clinical status of the patient or with overall survival (OS). As the systemic increase in target lesion size could be due to peritumoral inflammation that occurs when tumor lesions convert from cold to hot and new small lesions could be the result of inflammation of occult disease, it is difficult to distinguish tumor progression from pseudoprogression using CT scan imaging after experimental AlloStim® experimental treatment. Pseudoprogression after immunotherapy has been observed in patients with various tumor types and is thought to be due to transient immune cell infiltration into the tumor [ 33 ]. The phenomenon of pseudoprogression has led to modification of the RECIST 1.1 evaluation criteria [ 34 ]. The understanding that tumor growth by RECIST does not necessarily translate to disease progression in patients treated with immunotherapy has also led to the development of immune-related response criteria (irRC) to better surveil these patients [ 35 , 36 ]. However, it is still considered challenging to distinguish radiological progression from pseudoprogression and, consequently, to define the best management for these patients. In additional, a new category of “hyper-progression” and dissociated atypical responses have also been described after immunotherapy [ 37 ]. These issues have resulted in some subjects being prematurely removed from immunotherapy clinical trials [ 38 ]. Evidence from previous mCRC subjects treated with 3 cycles of experimental AlloStim® that presented with radiological progression at day 84 where simultaneous biopsy samples of target liver lesions were available suggested that the scans may over-estimate the actual tumor burden and supported that the tumor lesions converted from cold to hot (see Fig. 1). In the CheckMate 142 clinical trial, nivolumab (3 mg/kg) plus low-dose ipilimumab (1 mg/kg) provided durable clinical benefit, and a manageable safety profile in patients with previously treated dMMR/MSI-H metastatic CRC [ 39 ]. Due to the improved safety profile of the lower dose ipilimumab in this combination, we decided to test this regimen in a chemotherapy-refractory mCRC pMMR/MSS patient presenting with radiological progression after 3 cycles of AlloStim® experimental immunotherapy. We hypothesized that if the radiological progression was due to inflammation within the tumor lesions as seen previously (hot tumors), a short course of ICI immunotherapy should result in a rapid reduction in tumor burden, due to release of suppression of resident infiltrating effector immune cells. On the other hand, if the enlarged and increased number of lesions were due to true progression and no infiltrating effector immune cells were present, either no response or further progression would be expected to be observed. METHODS Case Presentation : A 69 yo Caucasian male presented in March 2011 with blood in the stool. The patient’s medical and social history was significant for type 2 diabetes, hypertension, hypercholesterolemia, atrial fibrillation (s/p ablation) and was a former 2 pack/day smoker. Upon workup, was found to have a rectal mass and subsequently underwent low anterior resection (LAR). 2 of 24 nodes were found to be positive for adenocarcinoma disease with initial staging of pT2N1b. The patient was treated with adjuvant FOLFOX and achieved a complete response (CR). In August 2014, disease recurred and he underwent transanal resection plus radiation therapy (XRT). In April 2016, a right inguinal node was identified as metastatic adenocarcinoma and he received additional XRT. In January 2022, he presented with enlarged retroperitoneal nodes and was treated with 22 cycles of FOLFIRI plus Avastin from January 2022 to November 2022. In January 2023, was treated again with FOLFOX, but developed a reaction to oxaliplatin. CT scan on April 26, 2023 showed appearance of innumerable bilateral pleural parenchymal lung nodules significantly increased in size and number from prior examination on March 8, 2023 and significant worsening of mediastinal and hilar lymphadenopathy consistent with progressive metastatic disease. No suspicious liver lesions were found and stable non-specific retroperitoneal adenopathy was also noted. Target lesions were identified in the lungs. The retroperitoneal disease was too small (< 15mm) to be included in the RECIST 1.1 evaluation. Patient consented on April 21, 2023 to received AlloStim® experimental immunotherapy as part of the STIMVAX Phase IIB clinical trial (NCT04444622). Eligible patients had histologically confirmed pMMR/MSS adenocarcinoma of the colon or rectum; received all available standard systemic therapies (fluoropyrimidines, oxaliplatin, irinotecan, and bevacizumab; cetuximab or panitumumab if RAS wild-type tumors); were aged 18 years or older; had adequate organ function; Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and measurable disease. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines after approval by a central institutional review board (IRB) and the ethics board at each institution where applicable. RESULTS The dosing and procedure schedule is shown in Table 1 and longitudinal changes in lung target lesions from CT scans are shown in Fig. 2. Three 28-day cycles of weekly AlloStim® immunotherapy were administered per protocol from May 9, 2023 to September 26, 2023. The CT scan comparison from baseline (April 26, 2023) to completion of the AlloStim® three cycles (October 10, 2023) demonstrated progressive disease (PD) by RECIST 1.1 evaluating target lesions in the lungs and retroperitoneum (sum of diameters of target lesions = + 73%) with increased non-target retroperitoneal adenopathy and innumerable enlarging and new thoracic nodules. Table 1 SCHEDULE OF PROCEDURES PROCEDURE ROUTE DATE CT Scan 26 Apr 23 AlloStim ID 09 May 23 AlloStim ID 16 May 23 AlloStim ID 23 May 23 AlloStim ID 30 May 23 AlloStim ID + IV 06 Jun 23 AlloStim ID 20 Jun 23 AlloStim ID 27 Jun 23 AlloStim ID 04 Jul 23 AlloStim ID 11 Jul 23 AlloStim ID/IV 15 Aug 23 AlloStim ID 29 Aug 23 AlloStim ID 5 Sep 23 AlloStim ID 12 Sep 23 AlloStim ID 19 Sep 23 AlloStim ID/IV 26 Sep 23 CT Scan 10 Oct 23 regorafenib 80 mg bid 11 Oct 23 − 22 Oct 23 regorafenib 80mg day 1/120mg day 2 23 Oct 23 − 12 Dec 23 Nivolumab 240 mg 23 Oct 23 Ipilimumab 1mg/kg 23 Oct 23 Nivolumab 240mg 13 Nov 23 Nivolumab 240mg 4 Dec 23 Ipilimumab 1mg/kg 4 Dec 23 Prednisone 60mg/day taper 12 Dec 23 − 7 Feb 24 CT Scan 8 Feb 24 From October 11, 2023 to October 23, 2023, a short, reduced dose of regorafenib was administered followed by a short course of nivolumab (240mg) and low dose ipilimumab (1mg/kg) administered between October 23, 2023 and December 4, 2023. No treatment was administered from December 12, 2023 and February 8, 2024 during which time a tapering dose of oral prednisone beginning at 60mg/day was administered for treatment of colitis (adverse effect from the combination ICI immunotherapy), a restaging CT scan was then obtained on February 8, 2024. The February 8, 2024 scan demonstrated a partial response (PR) by RECIST 1.1 criteria in comparison to the post-AlloStim® scan on October 10, 2023 (change in sum of diameters of target lesions = -48%) with the previously observed innumerable non-target thoracic lesions and retroperitoneal adenopathy uniformly decreased in size and number. Comparison to the April 26, 2023 baseline scan was read as stable disease (SD) with a 10% decrease in sum of diameters of target lesions. IL-12 and soluble heat shock protein (HSP)-70 levels were analyzed longitudinally (See Fig. 3). Serum IL-12 levels were evaluated to confirm whether maturation of dendritic cells to DC1 (IL-12+) occurred after AlloStim® immunotherapy. Serum levels of IL-12 levels were found to be negligible prior to AlloStim® administration. After AlloStim® dosing, IL-12 became detectable after the first cycle and remained detectible over the 3 cycles of AlloStim® administration, indicating an increase in mature DC1 dendritic cells. Soluble HSP-70 was also measured longitudinally to determine if immunological cell death occurred after AlloStim® administration, as HSP-70 is sequestered within tumor cells and detection in the serum would indicate release due to tumor cell membrane disruption. HSP-70 was found to be negligible at baseline, but was elevated throughout the experimental immunotherapy dosing. DISCUSSION Here we present a rare case of rapid tumor debulking response subsequent to a short course of combination ICI immunotherapy in a heavily pre-treated pMMR/MSS mCRC patient presenting with radiological progression after third-line experimental AlloStim® immunotherapy and a short course of low dose regorafenib. Here we consider the question whether the ICI immunotherapy alone or in combination with previous AlloStim®, or in combination with previous regorafenib alone, or with prior AlloStim® and regorafenib together was most likely responsible for eliciting this rare objective response in this cold tumor indication. It seems unlikely that the combination ICI immunotherapy could be solely responsible for the observed response. ICI-based regimens both as monotherapy [ 40 ] [ 41 ] or as combination therapies [ 42 ] have not previously shown any meaningful positive outcomes in pMMR/MSS colorectal cancers. For example, an initial phase II study assessed the efficacy of tremelimumab, a monoclonal antibody against CTLA4, in patients with treatment-refractory mCRC, which resulted in no improvement post-treatment [ 43 ]. Furthermore, two phase I studies of anti-PD-1 [ 44 ] and anti-PD-L1 [ 45 ] in previously-treated mCRC patients produced no responses. ICI regimens also failed as maintenance therapy after first line therapy in the MODUL study [ 46 ]. In general, ICI immunotherapy combining CTLA-4 and PD-L1 inhibitors have also shown very limited clinical benefit in patients with non-selected mCRC. A rare partial response (PR) (1/119) was reported in a randomized phase 2 clinical trial which evaluated the efficacy of combination durvalumab (anti-PD-L1) and tremelimumab (anti-CTLA-4) in patients with advanced refractory mCRC. In this study, 119 patients were assigned to the treatment group and 61 patients were assigned to best supportive care (BSC) alone. Patients in the treatment group received a median of 12 weeks of durvalumab and 12 weeks of tremelimumab [ 47 ], while in the present case only 5 weeks of ICI combination therapy was administered. The phase II KEYNOTE-016 trial was performed to evaluate the clinical efficacy of single agent pembrolizumab in patients with pMMR/MSS mCRC, dMMR/MSI-H mCRC and or dMMR/MSI-H non-CRC. No responses were noted in 18 patients in the pMMR/MSS mCRC group [ 48 ]. In a clinical study which included 59 pMMR/MSS mCRC patients treated with ICI beyond radiological progression by RECIST 1.1, no patient demonstrated subsequent radiographical tumor shrinkage at a median of 42 days [ 49 ]. It has been reported that a small subset (~ 2%) of patients with pMMR/MSS colorectal cancer with a mutation in POLE and POLD1 enzymes and those without liver metastases have a higher chance of a response to ICI immunotherapy [ 50 ]. While the patient in the present case did not have POLE or POLD1 mutations, no liver metastases were present. Therefore, it is possible this patient was more susceptible to ICI immunotherapy, but seems unlikely that the short course of combination ICI alone was solely responsible for the extensive tumor debulking observed. ICI strategies in combination with other drugs or procedures are under investigation, including evaluations of ICI in combinations with chemotherapy, radiotherapy, vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, and signal transducer and activation of transcription 3 (STAT3) inhibitors [ 51 ]. However, these combination approaches have yet to demonstrate any significant anti-tumor activity in the clinical setting [ 12 , 52 ], [ 53 ]. Could the short course of regorafenib alone or in combination with ICI immunotherapy be responsible for the objective tumor response observed in this case? Regorafenib is approved for third-line mCRC based on the results of the CORRECT trial which demonstrated only a 1.4 month increase in the median survival compared to a placebo control (6.4 months vs. 5.0 months) [ 54 ]. In the Phase II TEXCAN trial, no objective responses were reported in 35 mCRC patients after 2 months of treatment with regorafenib according to RECIST 1.1, Choi, and modified Choi [ 55 ]. Therefore, it seems unlikely that the prior short course of low dose regorafenib alone could be responsible for the rare objective response reported here. Regorafenib is a multi-kinase inhibitor that targets several receptor tyrosine kinases involved in angiogenesis and metastases (VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, TIE2, PDGFRs), oncogenesis (KIT, RET, RAF1), and tumor immunity (CSF1R). While regorafenib does not directly convert cold tumors to hot tumors, regorafenib is believed to possibly contribute to shifting the tumor microenvironment toward a more immune-responsive state. This constellation of mechanisms suggests that regorafenib could potentially be a combination partner for ICIs [ 56 ]. There are mixed results on the combination of regorafenib with ICI in clinical trials. Regorafenib in combination with PD-1 antibody as a third‐line mCRC therapy has been evaluated in several studies. For example, 24 patients with MSS mCRC were included in the REGONIVO study. In this study, regorafenib was administered at 80–160 mg once daily for 21 days on and 7 days off together with nivolumab at 3 mg/kg every 2 weeks. A 33.3% objective response rate was reported with this regimen [ 57 ]. However, this promising activity has not been observed in other studies. In a single site study, 18 mCRC patients treated with a combination of regorafenib and nivolumab, no objective responses were observed. In this study, 13 patients (69%) had progressive disease, and the median progression-free survival (PFS) was only 2 months. Four out of five patients in this study evaluated with stable disease (SD) occurred in patients without liver metastases, whereas a short disease stabilization was seen in 1 of 14 patients with history of liver metastases [ 58 ]. In another study in MSS mCRC patients, a combination of regorafenib and toripalimab, an anti-PD-1 ICI yielded an objective response rate of 15.2% (5 of 33 patients) with all (3 of 3) with lung-only metastasis responding [ 59 ]. In a retrospective study that involved 14 Chinese medical centers, a partial response rate of 5% (4 of 84 patients) was reported in MSS mCRC patients administered regorafenib combined with ICIs [ 60 ]. In a phase 2 study in patients from the USA with pMMR/MSS mCRC, regorafenib plus nivolumab yielded an objective response rate of 7%, with all responses observed in patients without liver metastases [ 61 ]. In this study, regorafenib was administered at 80 mg/day on a 3 weeks on/1 week off schedule and was increased to 120 mg/day if the 80 mg/day was well tolerated. Nivolumab was administered at 480 mg every 4 weeks. Based on these data, we cannot rule out the possibility that the regorafenib pre-treatment may have primed for responsiveness to the ICI immunotherapy in this pMMR/MSS mCRC patient that presented without liver metastases. However, in this case, the doses and frequencies of both regorafenib and of the combination ICI immunotherapy that were actually administered were significantly less that the doses administered in clinical trials where objective responses were observed. In addition, in the present case, corticosteroids (CS) were administered 6 weeks after start of ICI administration. In a retrospective single institution study, patients were evaluated in two cohorts based on timing of initiation of CS after initiation of ICI immunotherapy (≥ 2 months vs < 2 months). The administration of CS < 2 months after initiation of ICI immunotherapy was found to significantly hinder ICI efficacy [ 62 ]. Since regorafenib does not directly convert cold tumors to hot tumors, which is necessary for priming ICI responsiveness, and the doses and frequencies of both regorafenib and the ICI immunotherapy used in the present case were at sub-optimal therapeutic levels, combined with the early use of CS, we believe, while possible, it is unlikely that the regorafenib priming was responsible for the rare objective tumor response observed here and it is more likely that a combination that converted the cold tumors to hot was responsible. Therefore, we finally consider whether the experimental AlloStim® priming alone or in combination with regorafenib contributed to the ICI objective response. We hypothesized that if the restaging CT scan after AlloStim® immunotherapy reported as progressive disease (PD) by RECIST 1.1, was actually pseudoprogression due to ‘hot’ inflammation of the tumor lesions which would make them appear to be larger than the actual tumor burden, that ICI immunotherapy would elicit a rapid tumor debulking response due to resident infiltrating effector immune cell release from suppression. The present subject was negative for serum IL-12 at baseline. After three cycles of experimental AlloStim® immunotherapy the subject seroconverted to IL-12 positivity, supporting that the host immune system was modulated. We previously reported that IL-12 positivity correlated with long-term survival after AlloStim® immunotherapy [ 63 ]. IL-12 is an effector cytokine that promotes anti-tumor immunity by activating an effector Th1 response, which is required for the activation of cytotoxic T and NK cells [ 64 ]. IL-12 promotes production of IFN-γ which acts to upregulate PD-L1 in the tumor microenvironment (TME), which may make these tumors more susceptible to anti-PD-L1 ICI immunotherapy [ 65 – 67 ] . The presence of IL-12 can have many beneficial anti-tumor effects, including: increasing production of IFN-γ from NK and T cells [ 68 ]; stimulation of growth and cytotoxicity of activated NK cells and CD8 + and CD4 + T cells [ 69 ], shifting the Th1/Th2 balance in favor of the Th1 phenotype [ 70 ]; induction of antiangiogenic cytokine and chemokine production [ 71 ]; remodeling of the peritumoral extracellular matrix and tumor stroma [ 72 ], reprogramming of myeloid-derived suppressor cells [ 73 ], and increasing expression of MHC class I molecules necessary of cytolytic T-lymphocyte (CTL) recognition of tumor cells [ 74 ]. Soluble heat shock protein (HSP)-70 was also detected in the serum after AlloStim® administration. HSP-70 is a stress-inducible chaperone that is overexpressed within tumor cells, including CRC [ 75 ]. The finding of HSP-70 in serum suggests that tumor cells have been killed in a manner where the cell membrane is disrupted (immunological cell death), releasing the HSP along with danger signals into the tumor microenvironment. Hsp70 extracellular function is believed to be immunogenic and extracellular Hsp70 can serve as an adjuvant to activate the innate immune system [ 76 ] and can eventually lead to tumor-specific adaptive immunity [ 77 ]. Endogenous HSP chaperone all tumor cell antigens, including self- and neo-antigens. Tumors accumulate mutations that can cause tumor-specific neo-antigen expression. Since these neo-antigens are intracellular, they may not have been previously exposed to the immune system, as the tumors sequester these neoantigens. Thus the presence of soluble HSP-70 supports that AlloStim ® modified the TME in a manner that caused tumor lysis and release of chaperoned neoantigens. Exposure of tumor neoantigens to the immune system increases responsiveness to ICI [ 78 ]. The mechanism of action of AlloStim® is also consistent with the conclusion that an inflammatory cold to hot conversion occurred which caused the dramatic 73% increase in target lesion size by RECIST 1.1. The immune systems of patients with metastatic cancers are dysregulated resulting in a shift toward Th2 dominance [ 79 – 81 ]. AlloStim® experimental immunotherapy modulates the dysregulated immune systems of these patients to a Th1 dominance using a strategy of allo-priming [ 82 ]. The STIMVAX protocol incorporated a first series of intradermal injections of AlloStim®. The host rejection of the intentionally mis-matched AlloStim® cells shortly after administration results in increased titers of allo-specific Th1 and CTL cells, modulating the resident Th1/Th2 balance. These allo-specific cells elicited after intradermal injections are non-specifically activated by cytokine release after intravenous infusion of AlloStim® through a bystander activation mechanism [ 83 ]. Activated T-cells can extravasate and local inflammation attracts these cells into tissues and sites of inflammation, including tumors [ 84 ]. Thus, the intravenous infusion of AlloStim® after allo-priming can convert “cold” tumors into “hot” tumors with extensive infiltration of Th1/CTL memory cells, which could possibly account for the 73% increase in target lesion size. Inflamed tumor lesions can enlarge and appear as PD by RECIST 1.1. However, the putative anti-tumor mechanism leading to tumor debulking immunity may take several additional months before a radiological response can be detected and patients are often removed from the treatment protocols before a later assessment can be conducted. We hypothesized that based on the mechanism of action of AlloStim ® that radiological progression after 3 cycles likely represents a beneficial immune response that has primed the tumor lesions for an eventual debulking anti-tumor response. In order to support this hypothesis, we administered a short course of combination ICI immunotherapy. Since pMMR/MSS mCRC is known not to be responsive to ICI immunotherapy, we predicted that if a rapid tumor debulking response were observed, this would provide evidence supporting that the tumor lesions had been previously primed with infiltrating effector immune cells. CONCLUSION The available evidence makes it appear likely that AlloStim® played a role in eliciting the objective response observed after regorafenib and combination ICI immunotherapy. The rare objective response in this case provides support for further investigation of the combination of AlloStim ® combined with ICI immunotherapy with/or without regorafenib in ICI resistant patients. Abbreviations Abbreviation Definition bid twice a day BSC basic supportive care CRC colorectal cancer CS corticosteroid CSF1R colony stimulating factor receptor-1 CT computed tomography CTL cytolytic T-lymphocyte CTLA4 cytotoxic T-lymphocyte associated protein 4 CXCR3 C-X-C motif chemokine receptor-3 DC1 dendritic cell type 1 dMMR deficient DNA mismatch repair DNA deoxyribonucleic acid ECOG Eastern Cooperative Oncology Group FDA Food and Drug Administration FGFR1, epidermal growth factor receptor-1 FGFR2, epidermal growth factor receptor-2 FOLFIRI folinic acid, fluorouracil and irinotecan FOLFOX folinic acid, fluorouracil and oxaliplatin GVHD graft vs. host disease GVT graft vs. tumor ICI immune checkpoint inhibitor ID intradermal IFN interferon IL interleukin irRC immune related response criteria IV intravenous LAG lymphocyte activation gene LAR low anterior resection mCRC metastatic colorectal cancer MSI-H microsatellite instability high MSS microsatellite stable NK natural killer cell OS overall survival PBMC peripheral blood mononuclear cell PD progressive disease PD-1 programmed death receptor-1 PDGFR platelet derived growth factor receptor PD-L1 programmed death receptor-ligand 1 PFS progression free survival pMMR proficient DNA mismatch repair POLD1 polymerase delta-1 POLE polymerase epsilon PR partial response RAF1 Raf-1 proto-oncogene, serine/threonine kinase RAS rat sarcoma gene RECIST response evaluation in solid tumors s/p status post SD Stable Disease STAT3 Signal Transducer and Activator of Transcription 3 TH1 T-helper type 1 TH2 T-helper type 2 tid three times a day TIE2 TEK tyrosine kinase, endothelial TIL tumor infiltrating lymphocytes TMB tumor mutational burden TME tumor microenvironment USA United States of America VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor XRT radiation therapy Declarations Salus IRB, 2111 W. Braker Lane, Ste 100, Austin, TX 78758, serves as the central ethics committee for this study (ITL-032-MCRC3-STIMVAX). Informed consent was obtained from all individual participants included in the study. The patient consent form provided submission to publish results. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. ACKNOWLEDGEMENTS The authors wish to thank Axella Clinical Research (CRO) for assistance in coordinating data collection and research blood samples, Dr. Xiaochuan Yang and Eirini Topouzi for analyzing blood samples and assisting with preparation of figures. Dr. Elena Fingerut for managing the manufacturing of the study drug in accordance with good manufacturing practices and Thu Bui for managing blood donor collection pursuant to 21 CFR 1271 and the distribution of the frozen investigational product to the clinical site. Competing Financial Interests MHN is the inventor of the investigational AlloStim ® product and is the founder of Mirror Biologics, Inc. which sponsored the clinical study. AH and DG declare that they have no competing interests. Author Contributions MHN wrote the manuscript with input and review from other authors. AH is the principal investigator, conceived the design of the study and was responsible for implementing the protocol and for patient care. DW reviewed the longitudinal CT scans and determined the RECIST 1.1 conclusions and selected the CT scan images. Funding Mirror Biologics, Inc. is the sponsor of ITL-032-MCRC3-STIMVAX Phase IIB clinical trial and has funded the research. References Andre T, Shiu KK, Kim TW, Jensen BV, Jensen LH, Punt C, Smith D, Garcia-Carbonero R, Benavides M, Gibbs P, et al: Pembrolizumab in Microsatellite-Instability-High Advanced Colorectal Cancer. N Engl J Med 2020, 383: 2207-2218. 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Har-Noy M, Or R: Allo-priming as a universal anti-viral vaccine: protecting elderly from current COVID-19 and any future unknown viral outbreak. J Transl Med 2020, 18: 196. Paprckova D, Salyova E, Michalik J, Stepanek O: Bystander activation in memory and antigen-inexperienced memory-like CD8 T cells. Curr Opin Immunol 2023, 82: 102299. Newton P, O'Boyle G, Jenkins Y, Ali S, Kirby JA: T cell extravasation: demonstration of synergy between activation of CXCR3 and the T cell receptor. Mol Immunol 2009, 47: 485-492. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 01 Apr, 2024 Reviewers invited by journal 30 Mar, 2024 Editor assigned by journal 26 Mar, 2024 First submitted to journal 25 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4166800","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285666986,"identity":"f3b00c67-afdd-443d-bb7c-bbf038dd305b","order_by":0,"name":"Azriel Hirschfeld","email":"","orcid":"","institution":"Hirschfeld Oncology","correspondingAuthor":false,"prefix":"","firstName":"Azriel","middleName":"","lastName":"Hirschfeld","suffix":""},{"id":285666987,"identity":"996f9c67-d0b3-4406-9b32-76e2ac217043","order_by":1,"name":"Daniel Gurell","email":"","orcid":"","institution":"University Diagnostic Medical Imaging, PC","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Gurell","suffix":""},{"id":285666988,"identity":"8c18c98e-7682-4091-a7a8-4b775e3bf123","order_by":2,"name":"Michael Har-Noy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACAwhlw8AGohgbJIjWkka6lsMQirGBCIeZs5999uBj2/loPumzDx9X7rCQY2A/fHQDPi2WPenmhjPbbue28aUbG549I2HMwJOWdgOvww6ksUnzgrTwsLFJNrZJJDZI8Jjh13L+GUjLOVK03ADbcoAkLc/YJGecSwZpYTZsBPqFjaBfzqexSXwos8ud38PG+LBxR50cP/vhY3i1gAEjGxKHDacyFPCHOGWjYBSMglEwQgEAKvBCHnJCUxUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9342-9337","institution":"Mirror Biologics","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"","lastName":"Har-Noy","suffix":""}],"badges":[],"createdAt":"2024-03-26 03:59:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4166800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4166800/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54042747,"identity":"a19d6bf2-9569-4676-bbed-6d617af3959b","added_by":"auto","created_at":"2024-04-03 18:25:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-12 AND HSP70 SERUM LEVELS DURING ALLOSTIM ADMINISTRATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood samples were collected longitudinally from subjects in SST Tiger Top tubes. The tubes were spun at 3000 rpm and shipped overnight at 2-80C to the central lab facility where the serum was aseptically transferred to cryotubes and stored at -800C until analysis. For analysis, samples were diluted 1:2 and plated in triplicate on ELISA plates (R\u0026amp;D Systems) and incubated for 2-3h at RT. The plates were read on a Cytation 7 plate reader (Agilent BioTek) at 650nm absorbance. A standard curve was generated using known samples. Quantitative levels were determined by comparing absorbance values to the standard curve. The bar graph shows the mean +/- SE at each sample timepoint.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4166800/v1/2bac372adc94ae81987f6747.jpg"},{"id":54042748,"identity":"a6a4b7bd-6577-4878-80ea-a4f3a3842430","added_by":"auto","created_at":"2024-04-03 18:25:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205969,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal changes in two lung target lesions (orange arrows). The slices are adjusted to show the view with the measurement in the longest tumor diameter. Red circles indicate presence or absence of non-target lesions in the selected slices. An increase in size of non-target existing lesions and appearance of new non-target lesions seen on post-AlloStim October 10, 2023 compared to April 26, 2023 baseline. Elimination or reduction in size of the non-target lesions seen in the post-ICI February 8, 2024 scan. According to RECIST 1.1, the October 10, 2023 scan compared to April 26, 2023 is scored as PD. The February 8, 2024 scan compared to October 10, 2023 is scored as PR. The February 8, 2024 compared to April 26, 2023 is scored as SD.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4166800/v1/63bc2242b48b1ad9a3b6ef84.jpg"},{"id":54042746,"identity":"522a70be-42da-48b0-8d1c-287f34487d50","added_by":"auto","created_at":"2024-04-03 18:25:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71058,"visible":true,"origin":"","legend":"\u003cp\u003eMatched CT scans and biopsies of liver target lesions in two MSS mCRC subjects at baseline and at day 84 after AlloStim®. Subject #1 shows extensive tumor (red circle) on the periphery of areas of fibrosis without immune cell infiltration. The corresponding CT scan shows the target tumor. After 3 cycles of AlloStim®, the re-staging CT scan shows progressive disease. However, the corresponding biopsy shows areas of coagulative necrosis and immune cell infiltration. Subject #2 has almost completely solid tumor at baseline. The re-staging CT scan indicates progressive disease, however note the extensive peri-tumoral inflammation. The corresponding biopsy indicates large area of tumor necrosis and tumor admixed with immune cells.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4166800/v1/3bccef1095f7b20554c03be9.jpg"},{"id":54043924,"identity":"61be04e7-d6ad-4bfd-a2a4-b2d5cfb6c5ea","added_by":"auto","created_at":"2024-04-03 18:33:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2761344,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4166800/v1/fce7700a-fe05-41d9-9df2-cb1e63d6450f.pdf"}],"financialInterests":"","formattedTitle":"Objective Response after Immune Checkpoint Inhibitors in a Chemotherapy-Refractory\npMMR/MSS Metastatic Rectal Cancer Patient Primed with Experimental AlloStim®\nImmunotherapy.","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eImmune checkpoint inhibitor (ICI)-based regimens have not yet shown meaningful positive outcomes in proficient DNA mismatch repair/microsatellite stable (pMMR/MSS) metastatic colorectal cancers (mCRC). Here we report a rare objective response in a pMMR/MSS heavily pre-treated metastatic colorectal cancer (mCRC) patient subsequent to a short course of an immune checkpoint inhibitor (ICI) combination after first being primed with an experimental immunomodulatory cell therapy drug, AlloStim\u0026reg;, designed to convert immunologically \u0026lsquo;cold\u0026rsquo; tumors to \u0026lsquo;hot\u0026rsquo; tumors, and a short, low dose course of regorafenib.\u003c/p\u003e \u003cp\u003e The FDA has approved ICI drugs targeting CTLA-4, PD-1, PD-L1 and LAG-3 checkpoint molecules for a variety of solid tumor indications, including melanoma, renal, bladder, lung, gastric, gastroesophageal junction, hepatocellular carcinoma and head and neck cancers. However, ICIs have demonstrated only limited efficacy in mCRC.\u003c/p\u003e \u003cp\u003eAn anti-CTLA4 ICI, pembrolizumab, was approved in first line mCRC [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and also approved in combination with the anti-PD-1 ICI, nivolumab, for a small subset of mCRC patients that have deficient DNA mismatch repair/microsatellite instability-high (dMMR/MSI-H) status [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The dMMR/MSI-H subset constitutes only\u0026thinsp;~\u0026thinsp;5% of mCRC patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while the remaining\u0026thinsp;~\u0026thinsp;95% that present with pMMR/MSS status do not respond to ICI immunotherapy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost dMMR/MSI-H status tumors are considered immunologically \u0026lsquo;hot\u0026rsquo; tumors, while pMMR/MSS status tumors are considered to be immunologically \u0026lsquo;cold\u0026rsquo; [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hot tumors are characterized by an inflamed phenotype, including a high level of infiltrating T-cells and NK cells, an interferon-γ (IFN-γ) signature and upregulated PD-L1 expression, while cold tumors have an absence of tumor-infiltrating lymphocytes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile dMMR/MSI-H mCRC patients are more responsive to ICI therapy, approximately 50% are refractory [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Resistance to ICI responsiveness, regardless of MMR/MSI status, is correlated with tumor mutational burden (TMB) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Since somatic mutations can encode immunogenic neoantigens, high TMB is believed to be more likely to prime for infiltrating tumor-specific effector immune cells. Consistent with this, the dMMR/MSI-H patients that present with lower TMB values have been shown to be the non-responders, whereas patients with the highest TMB values tend to obtain benefit from ICI [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], particularly with anti-CTLA-4/PD-1 combination ICI immunotherapy[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePresent strategies for increasing the effectiveness of ICI immunotherapy in pMMR/MSS mCRC cold tumors include evaluating combinations with other therapeutic methods, such as chemotherapy, targeted therapy, and radiotherapy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, strategies to convert immunologically cold tumors to hot tumors are thought to be a fruitful line of investigation for increasing ICI efficacy in ICI refractory tumors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eAlloStim\u0026reg; is an experimental cellular immunotherapy drug designed to convert immunologically cold tumors to hot tumors by mirroring the graft vs. tumor (GVT) mechanism of allogeneic stem cell transplant procedures to create a host vs tumor (HVT) effect without graft vs. host disease (GVHD) toxicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. AlloStim\u0026reg; is currently being evaluated as a monotherapy in the STIMVAX Phase IIB open label clinical trial in third-line chemotherapy-refractory pMMR/MSS mCRC (NCT04444622). The primary end-point in this study is overall survival (OS) and an exploratory end-point is objective tumor response by RECIST 1.1.\u003c/p\u003e \u003cp\u003eAlloStim\u0026reg; is a living, allogeneic (\u0026ldquo;off-the-shelf\u0026rdquo;), non-genetically manipulated, activated Th1-like immune cell therapy derived from CD4\u0026thinsp;+\u0026thinsp;T-cell precursors isolated from the blood of healthy donors. The STIMVAX protocol provides for three monthly cycles of weekly AlloStim\u0026reg; administration (intradermal and intravenous) designed to modulate the immune character of cancer patients, including increase in circulating memory Th1/Th2 ratio [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], activation of circulating memory T cells and NK cells, causing their extravasation to tumor sites [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The anti-tumor effects of this immunomodulation are correlated with the establishment of an IFN-γ dominated microenvironment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe intravenous infusion and rejection of AlloStim\u0026reg; in primed allo-immune patients releases cytokines which serve to non-specifically activate memory cells and NK cells. Activated memory cells will extravasate and localize at sites of inflammation, including tumor lesions. This systemic tumor infiltration mechanism is believed to convert immunologically cold tumors to hot tumors. The modulation of the tumor microenvironment (TME) can also counter-regulate tumor-mediated immune suppression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInfiltrating memory T-cells and NK cells can produce intratumoral type I cytokines, including IL-12 and IFN-γ. These cytokines can act to: upregulate MHC-I on tumor cells making them susceptible to CD8\u0026thinsp;+\u0026thinsp;T-cell recognition; support immunological tumor cell death releasing neoantigens and danger signals into the TME [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]; cause maturation of dendritic cells that engulf and process chaperoned tumor neoantigens to DC1 (IL-12\u0026thinsp;+\u0026thinsp;CD80/86, MHC I and MHC II positive); and convert M2 macrophages to M1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTumor lysis that causes the release of internal chaperoned neoantigens into an inflammatory TME creates the conditions to support an in-situ vaccination mechanism [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In-situ vaccination involves immature dendritic cells processing the released chaperoned neoantigens to display on MHC I and MHC II, to mature to IL-12\u0026thinsp;+\u0026thinsp;DC1 and migrate to the draining lymph nodes, resulting in a patient-specific anti-tumor adaptive immune response [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn previous clinical studies, the inflammatory mechanism of AlloStim\u0026reg; almost always caused post-treatment CT scan images to be read as progressive disease (PD), with systemic increases in size of existing tumor lesions and often the appearance of new small lesions (especially in lungs). However, this PD determination did not always correlate with the clinical status of the patient or with overall survival (OS). As the systemic increase in target lesion size could be due to peritumoral inflammation that occurs when tumor lesions convert from cold to hot and new small lesions could be the result of inflammation of occult disease, it is difficult to distinguish tumor progression from pseudoprogression using CT scan imaging after experimental AlloStim\u0026reg; experimental treatment.\u003c/p\u003e \u003cp\u003ePseudoprogression after immunotherapy has been observed in patients with various tumor types and is thought to be due to transient immune cell infiltration into the tumor [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The phenomenon of pseudoprogression has led to modification of the RECIST 1.1 evaluation criteria [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The understanding that tumor growth by RECIST does not necessarily translate to disease progression in patients treated with immunotherapy has also led to the development of immune-related response criteria (irRC) to better surveil these patients [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, it is still considered challenging to distinguish radiological progression from pseudoprogression and, consequently, to define the best management for these patients. In additional, a new category of \u0026ldquo;hyper-progression\u0026rdquo; and dissociated atypical responses have also been described after immunotherapy [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These issues have resulted in some subjects being prematurely removed from immunotherapy clinical trials [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvidence from previous mCRC subjects treated with 3 cycles of experimental AlloStim\u0026reg; that presented with radiological progression at day 84 where simultaneous biopsy samples of target liver lesions were available suggested that the scans may over-estimate the actual tumor burden and supported that the tumor lesions converted from cold to hot (see Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eIn the CheckMate 142 clinical trial, nivolumab (3 mg/kg) plus low-dose ipilimumab (1 mg/kg) provided durable clinical benefit, and a manageable safety profile in patients with previously treated dMMR/MSI-H metastatic CRC [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Due to the improved safety profile of the lower dose ipilimumab in this combination, we decided to test this regimen in a chemotherapy-refractory mCRC pMMR/MSS patient presenting with radiological progression after 3 cycles of AlloStim\u0026reg; experimental immunotherapy.\u003c/p\u003e \u003cp\u003eWe hypothesized that if the radiological progression was due to inflammation within the tumor lesions as seen previously (hot tumors), a short course of ICI immunotherapy should result in a rapid reduction in tumor burden, due to release of suppression of resident infiltrating effector immune cells. On the other hand, if the enlarged and increased number of lesions were due to true progression and no infiltrating effector immune cells were present, either no response or further progression would be expected to be observed.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCase Presentation\u003c/span\u003e: A 69 yo Caucasian male presented in March 2011 with blood in the stool. The patient\u0026rsquo;s medical and social history was significant for type 2 diabetes, hypertension, hypercholesterolemia, atrial fibrillation (s/p ablation) and was a former 2 pack/day smoker. Upon workup, was found to have a rectal mass and subsequently underwent low anterior resection (LAR). 2 of 24 nodes were found to be positive for adenocarcinoma disease with initial staging of pT2N1b. The patient was treated with adjuvant FOLFOX and achieved a complete response (CR). In August 2014, disease recurred and he underwent transanal resection plus radiation therapy (XRT). In April 2016, a right inguinal node was identified as metastatic adenocarcinoma and he received additional XRT. In January 2022, he presented with enlarged retroperitoneal nodes and was treated with 22 cycles of FOLFIRI plus Avastin from January 2022 to November 2022. In January 2023, was treated again with FOLFOX, but developed a reaction to oxaliplatin.\u003c/p\u003e \u003cp\u003eCT scan on April 26, 2023 showed appearance of innumerable bilateral pleural parenchymal lung nodules significantly increased in size and number from prior examination on March 8, 2023 and significant worsening of mediastinal and hilar lymphadenopathy consistent with progressive metastatic disease. No suspicious liver lesions were found and stable non-specific retroperitoneal adenopathy was also noted. Target lesions were identified in the lungs. The retroperitoneal disease was too small (\u0026lt;\u0026thinsp;15mm) to be included in the RECIST 1.1 evaluation.\u003c/p\u003e \u003cp\u003ePatient consented on April 21, 2023 to received AlloStim\u0026reg; experimental immunotherapy as part of the STIMVAX Phase IIB clinical trial (NCT04444622). Eligible patients had histologically confirmed pMMR/MSS adenocarcinoma of the colon or rectum; received all available standard systemic therapies (fluoropyrimidines, oxaliplatin, irinotecan, and bevacizumab; cetuximab or panitumumab if RAS wild-type tumors); were aged 18 years or older; had adequate organ function; Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and measurable disease. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines after approval by a central institutional review board (IRB) and the ethics board at each institution where applicable.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe dosing and procedure schedule is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and longitudinal changes in lung target lesions from CT scans are shown in Fig.\u0026nbsp;2. Three 28-day cycles of weekly AlloStim\u0026reg; immunotherapy were administered per protocol from May 9, 2023 to September 26, 2023. The CT scan comparison from baseline (April 26, 2023) to completion of the AlloStim\u0026reg; three cycles (October 10, 2023) demonstrated progressive disease (PD) by RECIST 1.1 evaluating target lesions in the lungs and retroperitoneum (sum of diameters of target lesions\u0026thinsp;=\u0026thinsp;+\u0026thinsp;73%) with increased non-target retroperitoneal adenopathy and innumerable enlarging and new thoracic nodules.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSCHEDULE OF PROCEDURES\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePROCEDURE\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eROUTE\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDATE\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCT Scan\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e26 Apr 23\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e09 May 23\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e16 May 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e23 May 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e30 May 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u0026thinsp;+\u0026thinsp;IV\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e06 Jun 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e20 Jun 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e27 Jun 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e04 Jul 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e11 Jul 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID/IV\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e15 Aug 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e29 Aug 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e5 Sep 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e12 Sep 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e19 Sep 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAlloStim\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eID/IV\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e26 Sep 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eCT Scan\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e10 Oct 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eregorafenib\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e80 mg bid\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e11 Oct 23\u0026thinsp;\u0026minus;\u0026thinsp;22 Oct 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eregorafenib\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e80mg day 1/120mg day 2\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e23 Oct 23\u0026thinsp;\u0026minus;\u0026thinsp;12 Dec 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eNivolumab\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e240 mg\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e23 Oct 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eIpilimumab\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1mg/kg\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e23 Oct 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eNivolumab\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e240mg\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e13 Nov 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eNivolumab\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e240mg\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e4 Dec 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eIpilimumab\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1mg/kg\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e4 Dec 23\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003ePrednisone\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e60mg/day taper\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e12 Dec 23\u0026thinsp;\u0026minus;\u0026thinsp;7 Feb 24\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eCT Scan\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e8 Feb 24\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFrom October 11, 2023 to October 23, 2023, a short, reduced dose of regorafenib was administered followed by a short course of nivolumab (240mg) and low dose ipilimumab (1mg/kg) administered between October 23, 2023 and December 4, 2023. No treatment was administered from December 12, 2023 and February 8, 2024 during which time a tapering dose of oral prednisone beginning at 60mg/day was administered for treatment of colitis (adverse effect from the combination ICI immunotherapy), a restaging CT scan was then obtained on February 8, 2024.\u003c/p\u003e \u003cp\u003eThe February 8, 2024 scan demonstrated a partial response (PR) by RECIST 1.1 criteria in comparison to the post-AlloStim\u0026reg; scan on October 10, 2023 (change in sum of diameters of target lesions = -48%) with the previously observed innumerable non-target thoracic lesions and retroperitoneal adenopathy uniformly decreased in size and number. Comparison to the April 26, 2023 baseline scan was read as stable disease (SD) with a 10% decrease in sum of diameters of target lesions.\u003c/p\u003e \u003cp\u003eIL-12 and soluble heat shock protein (HSP)-70 levels were analyzed longitudinally (See Fig.\u0026nbsp;3). Serum IL-12 levels were evaluated to confirm whether maturation of dendritic cells to DC1 (IL-12+) occurred after AlloStim\u0026reg; immunotherapy. Serum levels of IL-12 levels were found to be negligible prior to AlloStim\u0026reg; administration. After AlloStim\u0026reg; dosing, IL-12 became detectable after the first cycle and remained detectible over the 3 cycles of AlloStim\u0026reg; administration, indicating an increase in mature DC1 dendritic cells.\u003c/p\u003e \u003cp\u003eSoluble HSP-70 was also measured longitudinally to determine if immunological cell death occurred after AlloStim\u0026reg; administration, as HSP-70 is sequestered within tumor cells and detection in the serum would indicate release due to tumor cell membrane disruption. HSP-70 was found to be negligible at baseline, but was elevated throughout the experimental immunotherapy dosing.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere we present a rare case of rapid tumor debulking response subsequent to a short course of combination ICI immunotherapy in a heavily pre-treated pMMR/MSS mCRC patient presenting with radiological progression after third-line experimental AlloStim\u0026reg; immunotherapy and a short course of low dose regorafenib. Here we consider the question whether the ICI immunotherapy alone or in combination with previous AlloStim\u0026reg;, or in combination with previous regorafenib alone, or with prior AlloStim\u0026reg; and regorafenib together was most likely responsible for eliciting this rare objective response in this cold tumor indication.\u003c/p\u003e \u003cp\u003eIt seems unlikely that the combination ICI immunotherapy could be solely responsible for the observed response. ICI-based regimens both as monotherapy [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] or as combination therapies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] have not previously shown any meaningful positive outcomes in pMMR/MSS colorectal cancers.\u003c/p\u003e \u003cp\u003eFor example, an initial phase II study assessed the efficacy of tremelimumab, a monoclonal antibody against CTLA4, in patients with treatment-refractory mCRC, which resulted in no improvement post-treatment [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, two phase I studies of anti-PD-1 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and anti-PD-L1 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] in previously-treated mCRC patients produced no responses. ICI regimens also failed as maintenance therapy after first line therapy in the MODUL study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn general, ICI immunotherapy combining CTLA-4 and PD-L1 inhibitors have also shown very limited clinical benefit in patients with non-selected mCRC. A rare partial response (PR) (1/119) was reported in a randomized phase 2 clinical trial which evaluated the efficacy of combination durvalumab (anti-PD-L1) and tremelimumab (anti-CTLA-4) in patients with advanced refractory mCRC. In this study, 119 patients were assigned to the treatment group and 61 patients were assigned to best supportive care (BSC) alone. Patients in the treatment group received a median of 12 weeks of durvalumab and 12 weeks of tremelimumab [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], while in the present case only 5 weeks of ICI combination therapy was administered.\u003c/p\u003e \u003cp\u003eThe phase II KEYNOTE-016 trial was performed to evaluate the clinical efficacy of single agent pembrolizumab in patients with pMMR/MSS mCRC, dMMR/MSI-H mCRC and or dMMR/MSI-H non-CRC. No responses were noted in 18 patients in the pMMR/MSS mCRC group [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In a clinical study which included 59 pMMR/MSS mCRC patients treated with ICI beyond radiological progression by RECIST 1.1, no patient demonstrated subsequent radiographical tumor shrinkage at a median of 42 days [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been reported that a small subset (~\u0026thinsp;2%) of patients with pMMR/MSS colorectal cancer with a mutation in POLE and POLD1 enzymes and those without liver metastases have a higher chance of a response to ICI immunotherapy [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. While the patient in the present case did not have POLE or POLD1 mutations, no liver metastases were present. Therefore, it is possible this patient was more susceptible to ICI immunotherapy, but seems unlikely that the short course of combination ICI alone was solely responsible for the extensive tumor debulking observed.\u003c/p\u003e \u003cp\u003eICI strategies in combination with other drugs or procedures are under investigation, including evaluations of ICI in combinations with chemotherapy, radiotherapy, vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, and signal transducer and activation of transcription 3 (STAT3) inhibitors [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, these combination approaches have yet to demonstrate any significant anti-tumor activity in the clinical setting [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCould the short course of regorafenib alone or in combination with ICI immunotherapy be responsible for the objective tumor response observed in this case?\u003c/p\u003e \u003cp\u003eRegorafenib is approved for third-line mCRC based on the results of the CORRECT trial which demonstrated only a 1.4 month increase in the median survival compared to a placebo control (6.4 months vs. 5.0 months) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the Phase II TEXCAN trial, no objective responses were reported in 35 mCRC patients after 2 months of treatment with regorafenib according to RECIST 1.1, Choi, and modified Choi [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, it seems unlikely that the prior short course of low dose regorafenib alone could be responsible for the rare objective response reported here.\u003c/p\u003e \u003cp\u003eRegorafenib is a multi-kinase inhibitor that targets several receptor tyrosine kinases involved in angiogenesis and metastases (VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, TIE2, PDGFRs), oncogenesis (KIT, RET, RAF1), and tumor immunity (CSF1R). While regorafenib does not directly convert cold tumors to hot tumors, regorafenib is believed to possibly contribute to shifting the tumor microenvironment toward a more immune-responsive state. This constellation of mechanisms suggests that regorafenib could potentially be a combination partner for ICIs [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are mixed results on the combination of regorafenib with ICI in clinical trials. Regorafenib in combination with PD-1 antibody as a third‐line mCRC therapy has been evaluated in several studies. For example, 24 patients with MSS mCRC were included in the REGONIVO study. In this study, regorafenib was administered at 80\u0026ndash;160 mg once daily for 21 days on and 7 days off together with nivolumab at 3 mg/kg every 2 weeks. A 33.3% objective response rate was reported with this regimen [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, this promising activity has not been observed in other studies.\u003c/p\u003e \u003cp\u003eIn a single site study, 18 mCRC patients treated with a combination of regorafenib and nivolumab, no objective responses were observed. In this study, 13 patients (69%) had progressive disease, and the median progression-free survival (PFS) was only 2 months. Four out of five patients in this study evaluated with stable disease (SD) occurred in patients without liver metastases, whereas a short disease stabilization was seen in 1 of 14 patients with history of liver metastases [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn another study in MSS mCRC patients, a combination of regorafenib and toripalimab, an anti-PD-1 ICI yielded an objective response rate of 15.2% (5 of 33 patients) with all (3 of 3) with lung-only metastasis responding [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In a retrospective study that involved 14 Chinese medical centers, a partial response rate of 5% (4 of 84 patients) was reported in MSS mCRC patients administered regorafenib combined with ICIs [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a phase 2 study in patients from the USA with pMMR/MSS mCRC, regorafenib plus nivolumab yielded an objective response rate of 7%, with all responses observed in patients without liver metastases [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In this study, regorafenib was administered at 80 mg/day on a 3 weeks on/1 week off schedule and was increased to 120 mg/day if the 80 mg/day was well tolerated. Nivolumab was administered at 480 mg every 4 weeks.\u003c/p\u003e \u003cp\u003eBased on these data, we cannot rule out the possibility that the regorafenib pre-treatment may have primed for responsiveness to the ICI immunotherapy in this pMMR/MSS mCRC patient that presented without liver metastases.\u003c/p\u003e \u003cp\u003eHowever, in this case, the doses and frequencies of both regorafenib and of the combination ICI immunotherapy that were actually administered were significantly less that the doses administered in clinical trials where objective responses were observed.\u003c/p\u003e \u003cp\u003eIn addition, in the present case, corticosteroids (CS) were administered 6 weeks after start of ICI administration. In a retrospective single institution study, patients were evaluated in two cohorts based on timing of initiation of CS after initiation of ICI immunotherapy (\u0026ge;\u0026thinsp;2 months vs\u0026thinsp;\u0026lt;\u0026thinsp;2 months). The administration of CS\u0026thinsp;\u0026lt;\u0026thinsp;2 months after initiation of ICI immunotherapy was found to significantly hinder ICI efficacy [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince regorafenib does not directly convert cold tumors to hot tumors, which is necessary for priming ICI responsiveness, and the doses and frequencies of both regorafenib and the ICI immunotherapy used in the present case were at sub-optimal therapeutic levels, combined with the early use of CS, we believe, while possible, it is unlikely that the regorafenib priming was responsible for the rare objective tumor response observed here and it is more likely that a combination that converted the cold tumors to hot was responsible.\u003c/p\u003e \u003cp\u003eTherefore, we finally consider whether the experimental AlloStim\u0026reg; priming alone or in combination with regorafenib contributed to the ICI objective response.\u003c/p\u003e \u003cp\u003eWe hypothesized that if the restaging CT scan after AlloStim\u0026reg; immunotherapy reported as progressive disease (PD) by RECIST 1.1, was actually pseudoprogression due to \u0026lsquo;hot\u0026rsquo; inflammation of the tumor lesions which would make them appear to be larger than the actual tumor burden, that ICI immunotherapy would elicit a rapid tumor debulking response due to resident infiltrating effector immune cell release from suppression.\u003c/p\u003e \u003cp\u003eThe present subject was negative for serum IL-12 at baseline. After three cycles of experimental AlloStim\u0026reg; immunotherapy the subject seroconverted to IL-12 positivity, supporting that the host immune system was modulated. We previously reported that IL-12 positivity correlated with long-term survival after AlloStim\u0026reg; immunotherapy [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-12 is an effector cytokine that promotes anti-tumor immunity by activating an effector Th1 response, which is required for the activation of cytotoxic T and NK cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. IL-12 promotes production of IFN-γ which acts to upregulate PD-L1 in the tumor microenvironment (TME), which may make these tumors more susceptible to anti-PD-L1 ICI immunotherapy [\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eThe presence of IL-12 can have many beneficial anti-tumor effects, including: increasing production of IFN-γ from NK and T cells [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]; stimulation of growth and cytotoxicity of activated NK cells and CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], shifting the Th1/Th2 balance in favor of the Th1 phenotype [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]; induction of antiangiogenic cytokine and chemokine production [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]; remodeling of the peritumoral extracellular matrix and tumor stroma [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], reprogramming of myeloid-derived suppressor cells [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], and increasing expression of MHC class I molecules necessary of cytolytic T-lymphocyte (CTL) recognition of tumor cells [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSoluble heat shock protein (HSP)-70 was also detected in the serum after AlloStim\u0026reg; administration. HSP-70 is a stress-inducible chaperone that is overexpressed within tumor cells, including CRC [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The finding of HSP-70 in serum suggests that tumor cells have been killed in a manner where the cell membrane is disrupted (immunological cell death), releasing the HSP along with danger signals into the tumor microenvironment. Hsp70 extracellular function is believed to be immunogenic and extracellular Hsp70 can serve as an adjuvant to activate the innate immune system [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] and can eventually lead to tumor-specific adaptive immunity [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Endogenous HSP chaperone all tumor cell antigens, including self- and neo-antigens. Tumors accumulate mutations that can cause tumor-specific neo-antigen expression. Since these neo-antigens are intracellular, they may not have been previously exposed to the immune system, as the tumors sequester these neoantigens. Thus the presence of soluble HSP-70 supports that AlloStim\u003csup\u003e\u0026reg;\u003c/sup\u003e modified the TME in a manner that caused tumor lysis and release of chaperoned neoantigens. Exposure of tumor neoantigens to the immune system increases responsiveness to ICI [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanism of action of AlloStim\u0026reg; is also consistent with the conclusion that an inflammatory cold to hot conversion occurred which caused the dramatic 73% increase in target lesion size by RECIST 1.1. The immune systems of patients with metastatic cancers are dysregulated resulting in a shift toward Th2 dominance [\u003cspan additionalcitationids=\"CR80\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. AlloStim\u0026reg; experimental immunotherapy modulates the dysregulated immune systems of these patients to a Th1 dominance using a strategy of allo-priming [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. The STIMVAX protocol incorporated a first series of intradermal injections of AlloStim\u0026reg;. The host rejection of the intentionally mis-matched AlloStim\u0026reg; cells shortly after administration results in increased titers of allo-specific Th1 and CTL cells, modulating the resident Th1/Th2 balance.\u003c/p\u003e \u003cp\u003eThese allo-specific cells elicited after intradermal injections are non-specifically activated by cytokine release after intravenous infusion of AlloStim\u0026reg; through a bystander activation mechanism [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Activated T-cells can extravasate and local inflammation attracts these cells into tissues and sites of inflammation, including tumors [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Thus, the intravenous infusion of AlloStim\u0026reg; after allo-priming can convert \u0026ldquo;cold\u0026rdquo; tumors into \u0026ldquo;hot\u0026rdquo; tumors with extensive infiltration of Th1/CTL memory cells, which could possibly account for the 73% increase in target lesion size.\u003c/p\u003e \u003cp\u003eInflamed tumor lesions can enlarge and appear as PD by RECIST 1.1. However, the putative anti-tumor mechanism leading to tumor debulking immunity may take several additional months before a radiological response can be detected and patients are often removed from the treatment protocols before a later assessment can be conducted.\u003c/p\u003e \u003cp\u003eWe hypothesized that based on the mechanism of action of AlloStim\u003csup\u003e\u0026reg;\u003c/sup\u003e that radiological progression after 3 cycles likely represents a beneficial immune response that has primed the tumor lesions for an eventual debulking anti-tumor response. In order to support this hypothesis, we administered a short course of combination ICI immunotherapy. Since pMMR/MSS mCRC is known not to be responsive to ICI immunotherapy, we predicted that if a rapid tumor debulking response were observed, this would provide evidence supporting that the tumor lesions had been previously primed with infiltrating effector immune cells.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe available evidence makes it appear likely that AlloStim\u0026reg; played a role in eliciting the objective response observed after regorafenib and combination ICI immunotherapy.\u003c/p\u003e \u003cp\u003eThe rare objective response in this case provides support for further investigation of the combination of AlloStim\u003csup\u003e\u0026reg;\u003c/sup\u003e combined with ICI immunotherapy with/or without regorafenib in ICI resistant patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"529\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDefinition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ebid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003etwice a day\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBSC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003ebasic supportive care\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003ecolorectal cancer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003ecorticosteroid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCSF1R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003ecolony stimulating factor receptor-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n 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\u003cp\u003e\u003cstrong\u003eFGFR1,\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eepidermal growth factor receptor-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFGFR2,\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eepidermal growth factor receptor-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFOLFIRI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003efolinic acid, fluorouracil and irinotecan\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFOLFOX\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003efolinic acid, fluorouracil and oxaliplatin\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGVHD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003egraft vs. host disease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGVT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003egraft vs. tumor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eICI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eimmune checkpoint inhibitor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eintradermal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIFN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003einterferon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003einterleukin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eirRC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eimmune related response criteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eintravenous\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003elymphocyte activation gene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003elow anterior resection\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003emCRC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003emetastatic colorectal cancer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMSI-H\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003emicrosatellite instability high\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMSS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003emicrosatellite stable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003enatural killer cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eOS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eoverall survival\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePBMC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eperipheral blood mononuclear cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eprogressive disease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eprogrammed death receptor-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePDGFR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eplatelet derived growth factor receptor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD-L1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eprogrammed death receptor-ligand 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePFS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eprogression free survival\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003epMMR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eproficient DNA mismatch repair\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePOLD1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003epolymerase delta-1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePOLE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003epolymerase epsilon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003epartial response\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRAF1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Raf-1 proto-oncogene, serine/threonine kinase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRAS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003erat sarcoma gene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRECIST\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eresponse evaluation in solid tumors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003es/p\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003estatus post\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eStable Disease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTAT3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignal Transducer and Activator of Transcription 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTH1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT-helper type 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTH2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eT-helper type 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003etid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003ethree times a day\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTIE2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTEK tyrosine kinase, endothelial\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTIL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003etumor infiltrating lymphocytes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTMB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003etumor mutational burden\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTME\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003etumor microenvironment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eUSA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnited States of America\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVEGF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003evascular endothelial growth factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVEGFR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003evascular endothelial growth factor receptor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.245746691871457%\"\u003e\n \u003cp\u003e\u003cstrong\u003eXRT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.75425330812854%\"\u003e\n \u003cp\u003e\u003cstrong\u003eradiation therapy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003eSalus IRB, 2111 W. Braker Lane, Ste 100, Austin, TX 78758, serves as the central ethics committee for this study (ITL-032-MCRC3-STIMVAX). Informed consent was obtained from all individual participants included in the study. The patient consent form provided submission to publish results. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Axella Clinical Research (CRO) for assistance in coordinating data collection and research blood samples, Dr. Xiaochuan Yang and Eirini Topouzi for analyzing blood samples and assisting with preparation of figures. Dr. Elena Fingerut for managing the manufacturing of the study drug in accordance with good manufacturing practices and Thu Bui for managing blood donor collection pursuant to 21 CFR 1271 and the distribution of the frozen investigational product to the clinical site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Financial Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMHN is the inventor of the investigational AlloStim\u003csup\u003e\u0026reg;\u003c/sup\u003e product and is the founder of Mirror Biologics, Inc. which sponsored the clinical study. \u0026nbsp;AH and DG declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMHN wrote the manuscript with input and review from other authors. AH is the principal investigator, conceived the design of the study and was responsible for implementing the protocol and for patient care. 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activation of CXCR3 and the T cell receptor.\u003c/strong\u003e \u003cem\u003eMol Immunol \u003c/em\u003e2009, \u003cstrong\u003e47:\u003c/strong\u003e485-492.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-medicine-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trmc","sideBox":"Learn more about [Translational Medicine Communications](http://transmedcomms.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/TRMC/default.aspx","title":"Translational Medicine Communications","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"immunotherapy, colorectal cancer, microsatellite stable, checkpoint inhibitor, microsatellite stable, mis-match repair proficient","lastPublishedDoi":"10.21203/rs.3.rs-4166800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4166800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Immune Checkpoint Inhibitor (ICI) immunotherapy is most effective in immune effector cell infiltrated ‘hot’ tumor lesions, such as occurs in deficient mismatch repair, microsatellite instability high (dMMR/MSI-H) colorectal cancer (CRC). However, most all metastatic CRC tumors are mismatch repair proficient/microsatellite stable (pMMR/MSS) ‘cold’ lesions, without significant immune cell infiltration, and are unresponsive to ICI. \u0026nbsp;AlloStim®, is an experimental, allogeneic immunomodulatory cell therapy designed to convert ‘cold’ metastatic tumor lesions to ‘hot’ inflamed lesions.\u0026nbsp; After AlloStim® immunotherapy, this cold to hot inflammatory mechanism can make it difficult to distinguish between pseudoprogression and actual progression on restaging CT scans, as inflamed metastatic lesions can appear larger and occult disease can appear as new small lesions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: To explore whether radiological progression after AlloStim® immunotherapy is due to immune-flare or disease progression, we administered a short course of a combination ICI therapy to a pMMR/MSS chemotherapy-refractory metastatic colorectal cancer patient enrolled in the StimVax Phase IIb clinical study that presented with radiological progression after AlloStim® immunotherapy. Our rationale was that an accelerated response to ICI should occur if the lesions were inflamed, while if the enlarged lesions were due to disease progression there would not be a response.\u0026nbsp; \u003cstrong\u003eResults\u003c/strong\u003e: Here we report a rapid, significant reduction in tumor burden in response to ICI administration in an AlloStim\u003csup\u003e®\u003c/sup\u003e primed pMMR/MSS mCRC patient with retroperitoneal and lung metastases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This rare objective response to ICIs in a pMMR/MSS mCRC patient supports further evaluation of the combination of AlloStim\u003csup\u003e®\u003c/sup\u003e with ICI immunotherapy in MSS mCRC and other cold or ICI refractory tumors.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTrial registration:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e National Library of Medicine (NLM) at the National Institutes of Health (NIH). Registered 22 June 2020, https://clinicaltrials.gov/study/NCT04444622\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Objective Response after Immune Checkpoint Inhibitors in a Chemotherapy-Refractory\npMMR/MSS Metastatic Rectal Cancer Patient Primed with Experimental AlloStim®\nImmunotherapy.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 18:25:16","doi":"10.21203/rs.3.rs-4166800/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-01T13:58:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-30T14:34:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-26T21:38:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Medicine Communications","date":"2024-03-25T23:59:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-medicine-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trmc","sideBox":"Learn more about [Translational Medicine Communications](http://transmedcomms.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/TRMC/default.aspx","title":"Translational Medicine Communications","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dee813d7-dc6f-478d-904d-2e4b44f5468a","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-30T20:27:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-03 18:25:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4166800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4166800","identity":"rs-4166800","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}