Off-tumor Toxicity Research Articles

Single-Cell Analysis of Target Antigens of CAR-T Reveals a Potential Landscape of "On-Target, Off-Tumor Toxicity".

Cellular immunotherapy represented by CD19-directed chimeric antigen receptor T (CAR-T) cells has achieved great success in recent years. An increasing number of CAR-T therapies are being developed for cancer treatment, but the frequent and varied adverse events, such as “on-target, off-tumor toxicity”, limit CAR-T application. Here, we identify the target antigen expression patterns of CAR therapies in 18 tissues and organs (peripheral blood mononuclear cells, bone marrow, lymph nodes, spleen, heart, ascending aortic tissue, trachea, lung, skin, kidney, bladder, esophagus, stomach, small intestine, rectum, liver, common bile duct, and pancreas) from healthy human samples. The atlas determines target antigens expressed on some normal cell types, which facilitates elucidating the cause of “on-target, off-tumor toxicity” in special tissues and organs by targeting some antigens, but not others. Moreover, we describe the target antigen expression patterns of B-lineage-derived malignant cells, acute myeloid leukemia (AML), and solid tumors. Overall, the present study indicates the pathogenesis of “on-target, off-tumor toxicity” during CAR therapies and provides guidance on taking preventive measures during CAR treatment.

Abstract P193: AMX-818, a novel prodrug HER2-XPAT T-cell engager (TCE) with potent T cell activation, proteolytic cleavage and efficacy in xenograft tumors, and wide safety margins in NHP (Non Human Primate)

Abstract Background: TCEs are effective in leukemias, but have been limited in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent cytokine release syndrome (CRS), including step-up dosing and complex designs, have had limited success due to toxicity and immunogenicity. AMX-818, HER2-XPAT, or XTENylated Protease-Activated T Cell Engager, is a prodrug TCE that exploits dysregulated protease activity present in tumors to expand the therapeutic index (TI). The core of the molecule (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core are two unstructured polypeptides (XTEN) that function as universal masks, sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, releasing a potent TCE with short T1/2. Methods: Nonclinical studies were conducted with HER2-XPAT or AMX-818, unmasked HER2-PAT, HER2-NoClvSite (no protease linkers) or the fluorescent labeled counterparts in vitro and in vivo xenografts and NHP. Results: The unmasked HER2-PAT demonstrated potent in vitro T cell cytotoxicity (EC50 1-2pM), target-dependent T cell activation and cytokine production by hPBMCs (human mononuclear cells). HER2-XPAT demonstrated 14,000-fold protection against killing of HER2 tumor cells, and minimal cytotoxicity against cardiomyocytes up to 1uM. In vivo, HER2-XPAT or AMX-818 induced complete tumor regressions (CRs) in HER2+ BT-474 tumors (similar to effect with equimolar doses of HER2-PAT), whereas HER2-NoClvSite lacked efficacy, supporting the need for protease cleavage for activity. HER2-XPAT was also highly efficacious in the HER2 low-expressing HT-55 colorectal model. Labeled XPATs with the same protease cleavage sites in 9 different in vivo xenograft and PDX models demonstrated preferential unmasking into cleaved products in tumors in contrast to healthy organs, with average ~20% of drug in tumor cleaved to the fully active TCE form. In NHPs, AMX-818 caused early T-cell margination as low as 2mg/kg, but has been dose-escalated safely up to 42mg/kg (MTD) with no CRS. Doses of 0.5 to 42 mg/kg demonstrated dose proportional increases in HER2-XPAT exposure, with low to negligible levels of cleavage products present in systemic circulation. Continuous infusion of unmasked HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d but was tolerated at 0.2 mg/kg/d, providing AMX-818 with ~450-fold improvement in safety compared to HER2-PAT. Repeat-dose GLP toxicology studies have demonstrated that the highest dose evaluated (6 mg/kg) was the NOAEL, suggesting that HER2-XPAT has a wide TI based on predicted human PK and predicted efficacious exposures. Conclusions: AMX-818 is a potent, protease-activated prodrug TCE exhibiting preferential tumor unmasking, with minimal systemic unmasking and no CRS at high doses in NHP. AMX-818 represents a promising solution to widen the TI for TCE activity in HER2-high and HER2-low expressing tumors. Citation Format: Milton To, Pete Yeung, Michael Fox, Mikhail Hammond, Fiore Cattaruzza, Ayesha Nazeer, Caitlin Koski, Lucas Liu, Sina Khorsand, Deena Rennerfeldt, Kari Morrissey, Zachary Lange, Ming Dong, Sharon Lam, Mika K. Derynck, Bryan A. Irving, Volker Schellenberger. AMX-818, a novel prodrug HER2-XPAT T-cell engager (TCE) with potent T cell activation, proteolytic cleavage and efficacy in xenograft tumors, and wide safety margins in NHP (Non Human Primate) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P193.

Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells

BackgroundChimeric antigen receptor (CAR) T cells have been successfully used in tumor immunotherapy due to their strong antitumor responses, especially in hematological malignancies such as B cell acute lymphoid leukemia....

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients.Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs).Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p<0.05) or CD33 (p<0.01), and exhibited >80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p<0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p<0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p<0.05) and MV4-11 (p<0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape.Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p<0.01) or 50% (iCAR#2, p<0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p<0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis.Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects. DisclosuresNo relevant conflicts of interest to declare.

Multiplex Engineering of Human CD34+ HSPCs Enables Dual Gene Knock-out While Maintaining High Engraftment Potential and Safety

Genome engineering of allogeneic hematopoietic stem cell transplants (HSCT), in which leukemia-specific cell surface antigens are removed by editing from the donor graft, may provide high therapeutic index and safety in combination with post-transplant targeted therapeutics. This strategy allows the therapeutics to specifically target leukemic cells by sparing the gene-edited graft, and thereby enable the next generation of HSCTs for treatment of acute myeloid leukemia (AML). So far, no single target antigen is known to be expressed in 100% of AML blasts, leukemic stem cells, or even in affected patients. Therefore, use of combinatorial therapies targeting multiple antigens may represent a potential paradigm shift in the standard of care for AML. Multiplex editing of HSCT in which two or more target antigens are removed from the same cell would enable combinatorial targeting while simultaneously protecting the engineered graft from on-target, off-tumor toxicity. Here we report proof-of-concept data that multiplex editing with two independent CRISPR/Cas9 ribonucleoproteins (RNPs) targeting genetic loci on chromosomes 11 and 19 robustly maintains long-term in vivo engraftment potential with persistent gene edits at both target sites, with minimally detectable translocations in bone marrow samples 16 weeks after engraftment in NSG mice. The overall viability of dual-edited input CD34+ HSPCs post gene editing was >75% and the proportion of CD34+CD38-CD45RA-CD90+CD49f+ putative long-term hematopoietic stem cells (LT-HSCs) in input cells were similar (1-2%) between unedited and multiplex-edited cells. In vivo engraftment potential of multiplex-edited cells was maintained long-term, with average chimerism of hCD45+ cells in mice transplanted with dual knock-out (KO) HSPCs similar to those transplanted with unedited HSPCs (53.36±11.80% versus 47.45±18.03%, respectively). Dual KO cells also maintained normal differentiation potential as judged by multilineage reconstitution in vivo and in vitro colony forming unit (CFU) assay, indicating the dual RNP delivery process does not lead to any observable impact on the multipotency of the engineered graft. Quantification of on-target editing by multiplexed NGS amplicon sequencing revealed no reduction in total editing between dual KO input and bone marrow cells, indicating the gene modifications in dual engineered cells persist post-engraftment.‡equal contribution, Nipul Patel and Michael A. Pettiglio DisclosuresPettiglio: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Pimentel: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Kapuria: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ghodssi: Vor Biopharma: Current Employment, Current holder of stock options in a privately-held company. Isik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Falla: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Amunugama: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lisle: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Wang: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lydeard: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hazelbaker: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Paik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

Interim Results from the First Clinical Study of CC-95251, an Anti-Signal Regulatory Protein-Alpha (SIRPα) Antibody, in Combination with Rituximab in Patients with Relapsed and/or Refractory Non-Hodgkin Lymphoma (R/R NHL)

Interim Results from the First Clinical Study of CC-95251, an Anti-Signal Regulatory Protein-Alpha (SIRPα) Antibody, in Combination with Rituximab in Patients with Relapsed and/or Refractory Non-Hodgkin Lymphoma (R/R NHL)

Developing a Safer Anti-CD44v6 Chimeric Antigen Receptor T Cell Against Hematological Cancers By Mitigating on-Target Off-Tumor Toxicity

Adoptively transferred chimeric antigen receptor (CAR) T cells have shown significant promise in targeted immunotherapy against hematological tumors, despite concerns with safety and antigen escape. The CD44 adhesion molecule, which binds components of ECM such as collagen and hyaluronan, has been implicated in growth and survival as well as metastasis of tumor initiating stem cells (TSCs). A splice variant of CD44, CD44v6 is broadly expressed on malignant tumors including AML, CML and multiple myeloma (MM), where it aids tumor migration and predicts poor disease prognosis 1. Moreover, the expression of CD44v6 on healthy tissues including keratinocytes, presents a significant challenge in ‘on-target, off-tumor’ toxicity 2,3. We have developed a second generation, anti-CD44v6 CAR T cell (CD44v6CAR) that mediates potent cytotoxic and effector function against AML and MM in-vitro. Additionally, we observed lesser potency of our CD44v6CAR T cells against MM in comparison with AML in-vivo. Interestingly, higher levels of soluble CD44v6 antigen were also detected in mice engrafted with MM.1S compared to THP1. Possibly, the presence of soluble CD44v6 in mouse serum culminated in reduced anti-tumor activity against MM in-vivo, most likely due to CAR scFv blockade with antigen. Here, we provide an alternative strategy for improving the CD44v6 CAR T cell therapy and mitigating on-target off-tumor toxicity using CD44v6 CAR T cells that secrete a soluble protease-susceptible version of the CAR-expressed CD44v6 scFv that will block CAR binding in healthy tissue, but will be cleaved by cancer-specific proteases in the tumor site, allowing for CAR T cell binding and activation (Figure 1). We leveraged the presence of matrix metalloproteinase (MMP)-2, which is significantly overexpressed in multiple myeloma tumor microenvironment 4 to develop a modified CAR T construct (sCD44v6CAR) with engineered MMP-2 cut site in the linker between the heavy and light chains of the soluble CD44v6scFv. Kinetic analysis with varying MMP-2 concentration and digestion time showed moderate proteolytic susceptibility of our scFv construct, with digestion efficiency increasing in a dose-dependent manner. Furthermore, our engineered protease-liable scFv demonstrated higher affinity for CD44v6 antigen binding in titration assays compared to CAR scFv suggesting that engineered scFv are able to 1) bind and neutralize soluble antigen and 2) bind antigen on healthy tissues with high affinity to mitigate off-tumor toxicity. As expected, both conventional (CD44v6CAR) and modified sCD44v6 CAR T cells showed effective cytotoxicity against AML in vitro. Interestingly, cytotoxic activity against MM.1S using the modified T cells (sCD44v6CAR) was significantly suppressed, likely resulting from secretion of soluble scFv. The addition of recombinant MMP-2 in co-culture assays cleaved soluble scFvs, rescuing CAR-mediated tumor killing. Taken together, the data confirms our proof-of-concept hypothesis and highlights the protective capacity of engineered sCD44v6CAR T cells, with its ability to potentially neutralize off-target toxicity and improve anti-MM activity in future studies, which has impact on the CAR T cell therapy as a general strategy.Figure 1: Schematic representation of CAR T Cell mitigating off-disease recognition. Presence of protease-susceptible linker in soluble scFv is efficiently cleaved by tumor-specific proteases in tumor microenvironment enabling CAR binding and activation. Lack of specific proteases in healthy tissues leads to high affinity, soluble scFv-target binding and effective blocking.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

A Phase 1, Open-Label, Dose-Escalation Study of the Safety and Efficacy of Anti-CD38 Antibody Drug Conjugate (STI-6129) in Patients with Relapsed or Refractory Multiple Myeloma

Background: With the advent of several new agents in the treatment of multiple myeloma, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and anti-CD38 monoclonal antibodies (mAb), the overall survival (OS) has significantly improved in the past two decades. However, most patients become refractory to currently available therapies along the disease trajectory. The median OS in triple-class-refractory patients (i.e., anti-CD 38 mAb, PIs, and IMiDs) is 6 months (Gandhi et al. Leukemia. 2019). Although BCMA-targeted therapies are a major advance in such patients, none of them are curative thus far, with the median progression-free survival (PFS) ranging from 3 months in BCMA-antibody drug conjugates (ADCs) to approximately 1 year in BCMA chimeric antigen receptor T-cell (CAR T-cell) therapies. Furthermore, unique toxicities of BCMA-targeted agents such as ocular toxicity (ADCs), cytokine release syndrome, or neurotoxicity (CAR Ts and BiTEs) may preclude their use in many patients. Hence, there is a critical unmet need in patients with triple-class-refractory myeloma.The STI-6129 ADC is produced by conjugating STI-5171, a fully human anti-CD38 mAb, with a covalently bound tubulin inhibitor, duostatin 5.2 (DUO-5.2), using a proprietary linker technology with a 3:1 drug-antibody ratio. STI-6129 binds to different CD38 epitopes than daratumumab. Upon binding to CD38, STI-6129 ADC is internalized by the cancer cell and undergoes lysosomal degradation, releasing DUO-5.2. This in turn leads to G2-phase cell cycle arrest, followed by caspase 3/7-dependent apoptosis and cell death. In studies on cynomolgus monkeys, the serum level of DUO-5.2 remained undetectable at all doses except at the highest dose of 4.5 mg/kg, indicating low likelihood of off-target toxicity. In vitro studies of primary human plasma cells, human tumor models, and animal xenograft models have demonstrated target elimination of CD38-positive human plasma cells by STI-6129. Importantly, STI-6129 has demonstrated cytotoxic activity in human multiple myeloma cells isolated from daratumumab-refractory patients (Figure 1). Hence, further investigation on clinical activity of STI-6129 is warranted.Study Design: The 38-ADC-RRMM-101 study is a two-stage, multicenter, open-label, dose-finding phase 1/2 trial. The phase 1 trial is designed to identify the recommended phase 2 dose (RP2D) of STI-6129 by assessing safety, preliminary efficacy, and pharmacokinetics in patients with relapsed/refractory (RR) myeloma. The phase 2a stage of the trial will be a single-arm study to investigate the efficacy of STI-6129 in an expansion cohort of RR myeloma patients. Up to 25 patients will be enrolled in the phase 1 stage and 30 in the phase 2 stage of the study. The key inclusion criteria are the following: (a) RR myeloma with at least 3 prior lines of therapy in addition to being refractory to at least 1 PI, 1 IMiD, and 1 anti-CD 38 mAb; (b) measurable disease. The key exclusion criteria are: (a) receipt of the last dose of any anti-CD mAb within 90 days; (b) grade ≥3 neuropathy or grade 2 neuropathy with pain; (c) current history of CTCAE grade 3 muscle paresis, eyelid conditions, glaucoma, or any other ocular disorder that is CTCAE grade 2; (d) estimated creatinine clearance <60 ml/min; (e) left ventricular ejection fraction<40%. The primary endpoint of phase 1 stage of the study is safety, particularly any dose limiting toxicities. The primary endpoint of the phase 2 part of the study is overall response rate as per IMWG criteria. The dosing cohorts in the dose escalation phase will range from 0.25 mg/kg to 3.68 mg/kg. Given the potential for on-target off-tumor toxicity due to expression of CD38 on non-clonal plasma cells and other hematopoietic cells, blood counts and immunoglobulin levels will be closely monitored. While neurotoxicity or ocular toxicity were not observed with STI-6129 in animal models, including non-human primates, such event will be considered as adverse events of special interest (AESI) and comprehensive neurology and ocular (including slit lamp) examinations will be performed at baseline and study completion, and at any AESI. STI-6129 will be administered intravenously once in a 4-week cycle, with the intention being to treat patients until disease progression or unacceptable toxicity. [Display omitted] DisclosuresYan: Sorrento Therapeutics: Current Employment. Royal: Sorrento Therapeutics: Current Employment.

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

CD123 and CD33 Co-Targeting By Balanced Signaling on CAR-CIK Cells Reduces Potential Off-Target Toxicity While Preserving the Anti-Leukemic Activity of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated a high therapeutic potential in B-acute lymphoblastic leukemia, but translation in AML is limited by the absence of an ideal tumor-specific antigen. CD123 (known as IL-3 receptor alfa) and CD33 satisfy several features of ideal target antigens as they are expressed in almost all AML patients (mainly overexpressed in NPM1 and FLT-3 mutated AML), and conserved at disease relapse . Despite that, CD33 expression on hemopoietic stem/progenitor cells (HSPC) induced prolonged myelotoxicity after anti-CD33 CAR-T cell therapy and CD123 expression on endothelium was responsible for severe capillary leak syndrome after anti-CD123 CAR therapy. With the aim to improve selectivity for CD123+/CD33+ AML cells while minimizing toxicity against healthy cells, we probe a dual targeting model by Cytokine Induced Killer (CIK) cells co-expressing a first generation low affinity anti-CD123 IL-3 zetakine (IL3z.CAR) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR). This trans-signaling strategy could allow: 1) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; 2) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; 3) full CAR-CIK activation only against double expressing CD123 + / CD33 + leukemic cells.Fresh and frozen peripheral blood mononuclear cells were transduced with retroviral vectors during the CIK cell differentiation process . The functional activity of CAR-CIK cells was assessed in vitro by means of short- and long- term cytotoxicity and cytokine production assays upon challenge with different CD123/CD33 positive AML cell lines (THP-1 and KG-1) or with a CD123 positive human endothelial cell line (TIME). To assess the myelotoxicity against HSPC, CD34+ cells were sorted after 24h co-culture with CAR-CIK cells and mixed with methylcellulose-based semisolid medium to evaluate Colony Forming Units (CFU) after 14 days through an automated imaging and counting system for hematopoietic colonies (Stemvision). The in vivo anti-leukemic activity was evaluated by monitoring luciferase-expressing KG1 AML cell line growth over time in NSG mice treated with 3 infusions of all different CAR-CIK conditions.Dual CAR-CIK cells (IL-3z.CAR/CD33.CCR) display a potent and specific in vitro anti-leukemic efficacy against all the AML cell lines tested, compared to single targeting IL-3z.CAR and non-transduced CIK cells. To further minimize toxicities against healthy cells exploiting differences in CD123 antigen density between leukemic and healthy cells, we generated a low affinity dual CAR decreasing the IL3z.CAR binding affinity to CD123, by site-directed mutagenesis. Low affinity dual CAR-CIK cells show irrelevant cytotoxicity against the TIME endothelial cell line, comparable to non-transduced CIK cells, while preserving the efficacy against THP1 and KG1 AML cell lines. As a confirm of the reduced reactivity against endothelial cells, low affinity dual CAR-CIK cells produce the same levels of IL-2 and IFNγ either alone or in the presence of TIME cell line. Low affinity dual CAR-CIK cells also preserve the in vitro CFU-E, CFU-GM and CFU-GEMM colony forming capacity as compared to wild type (wt) dual CAR-CIK and wt IL-3z.CAR-CIK cells, highlighting the possibility to find a therapeutic window to minimize toxicity on healthy cells. The anti-leukemic activity of low affinity dual CAR-CIK cells has been confirmed also in vivo, with a significant suppression of leukemic growth against KG1 AML cell line. Mice treated with 3 infusions of low affinity dual CAR-CIK cells exhibited a significant better anti-leukemic profile, when compared to IL-3-single targeting CAR-CIK cells in terms of tumor growth and overall survival.These preclinical data demonstrate a powerful antitumor efficacy mediated by low affinity dual targeting IL-3z.CAR/CD33.CCR CIK cells against AML targets without any relevant in vitro toxicity on HSPC and endothelial cells, offering a proof-of-concept strategy to increase selectivity for CD123+/CD33+ AML cells whilst reducing the risk of “on-target off-tumor toxicity”. DisclosuresPerriello: Novartis: Other: Advisory Board. Martelli: Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals: Honoraria. Falini: Rasna Therapeutics: Honoraria. Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria.

Implementing Logic Gates for Safer Immunotherapy of Cancer.

Targeting solid tumors with absolute precision is a long-standing challenge in cancer immunotherapy. The identification of antigens, which are expressed by a large fraction of tumors of a given type and, preferably, across various types, but not by normal cells, holds the key to developing safe, off-the-shelf immunotherapies. Although the quest for widely shared, strictly tumor-specific antigens has been the focus of tremendous effort, only few such candidates have been implicated. Almost all antigens that are currently explored as targets for chimeric antigen receptor (CAR) or T cell receptor (TCR)-T cell therapy are also expressed by healthy cells and the risk of on-target off-tumor toxicity has remained a major concern. Recent studies suggest that this risk could be obviated by targeting instead combinations of two or more antigens, which are co-expressed by tumor but not normal cells and, as such, are tumor-specific. Moreover, the expression of a shared tumor antigen along with the lack of a second antigen that is expressed by normal tissues can also be exploited for precise recognition. Additional cues, antigenic or non-antigenic ones, which characterize the tumor microenvironment, could be harnessed to further increase precision. This review focuses on attempts to define the targetable signatures of tumors and assesses different strategies employing advanced synthetic biology for translating such information into safer modes of immunotherapy, implementing the principles of Boolean logic gates.

In This Issue

See article, p. 568.Hematologic malignancy patients are highly susceptible to severe coronavirus disease-19 (COVID-19) illness and are a high-priority group for vaccination to mitigate COVID-19–related morbidity and mortality. Chung et al. find differential COVID-19 vaccine humoral responses between hematologic malignancy subtypes and disease-specific therapies across patient cohorts. Hematologic malignancy patients undergoing active therapy have baseline humoral responses that are further suppressed. Importantly, antibody titers alone are an imperfect measure of immunity, as many patients with positive anti–SARS-CoV-2 antibody titers have insufficient neutralizing capacity. These findings together provide important metrics for optimizing the clinical care of this vulnerable population.See article, p. 577.Cellular therapies, including allogeneic and autologous hematopoietic cell transplant (allo-HCT, auto-HCT) and chimeric antigen receptor T-cell therapy (CAR T), render patients severely immunocompromised for extended periods after therapy. Tamari et al. define predictors of response to COVID-19 vaccine to nominate important factors for optimizing response to vaccination. Type of cellular therapy, timing of vaccination post–cellular therapy, and immune recovery post–cellular therapy predict response. Many patients with positive, yet low, anti–SARS-CoV-2 antibody titers demonstrate low neutralizing capacity, highlighting the limitations of using antibody titer as the only proxy for immunity.See article, p. 586.Pediatric acute leukemias have historically been classified by immunophenotyping followed by genomic subclassification. Patients are categorized into T, B, myeloid, or mixed-lineage disease for the purpose of general treatment approach, and these regimens are then further tailored in intensity and/or targeted therapeutics when applicable. Fornerod et al. apply an integrated analysis of gene expression and mutation profiling to a cohort of pediatric patients with acute leukemias that have myeloid features. This reveals a subset of patients with shared mutational composition and gene expression that spanned three immunophenotypes: T, myeloid, and mixed-lineage acute leukemias. The authors further identify patients with the same mutations but with differential gene expression profiles and outcomes. These results suggest acute leukemia classification requires more comprehensive, sequencing-based diagnostic methods beyond flow cytometry.See article, p. 600.Waldenstrom macroglobulinemia (WM) is characterized by outgrowth of mature IgM+ B cells with MYD88 mutations. Kaushal et al. show that WM lesions originate in the presence of several alterations in the immune microenvironment, including aberrant nonmalignant extrafollicular B-cell expansion, myeloid inflammation, and WM-specific T-cell immune responses. The characteristic MyD88 mutations originate in early lymphoid progenitors prior to expansion of the malignant clone. These findings suggest a new model for the origin of WM.See article, p. 616.Overall outcomes are dismal in relapsed/refractory AML (R/R AML), and therapeutic options are limited. In this phase II study, Zeidner et al. investigate the safety, feasibility, and clinical activity of immune checkpoint blockade with pembrolizumab after high-dose cytarabine (HiDAC) chemotherapy in R/R AML. The addition of pembrolizumab to HiDAC leads to an overall complete remission rate of 38%, with encouraging clinical outcomes and self-limiting immune-related adverse events. Multifaceted immunogenomic profiling before and after therapy reveals putative biomarkers of response to pembrolizumab, warranting further investigation.See article, p. 630.Inhibitors of Bruton tyrosine kinase (BTKi) block pathogenic B-cell receptor (BCR) signaling, improving outcomes for patients with lymphoid malignancies. Nonetheless, resistance to BTK therapy is a frequent clinical problem that has previously been ascribed to genetic alterations. Shaffer et al. find a primary, early mechanism of BTKi resistance involving epigenetic reprogramming of the malignant cell. Specifically, the epigenetic upregulation of the small GTPase RAC2 increases its interaction with, and activation of, PLCγ2. This RAC2-dependent activation circumvents the requirement for BTK to phosphorylate PLCγ2 to trigger the prosurvival NF-κB pathway. Clinically available drugs are identified that can kill BTKi-resistant cells.See article, p. 648.Clinical translation of CAR T-cell therapy for AML has been limited by the lack of optimal CAR targets and the risk of on-target, off-tumor toxicity. Richards et al. report a novel anti-AML CAR T-cell therapy targeting CD93 and demonstrate antileukemic activity without toxicity to critical hematopoietic stem and progenitor cells. To mitigate tissue-specificity concerns, next-generation, NOT-gated CD93 CAR T cells were engineered. Transcriptomic analysis of AML and endothelial cells demonstrates a promising target identification strategy for further advancement of this technology.

In This Issue

In This Issue

Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV.

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.

Conditioned CAR-T cells by hypoxia-inducible transcription amplification (HiTA) system significantly enhances systemic safety and retains antitumor efficacy

BackgroundHypoxia is a striking feature of most solid tumors and could be used to discriminate tumors from normoxic tissues. Therefore, the design of hypoxia-conditioned Chimeric Antigen Receptor (CAR) T cells...

Rethinking mechanisms of neurotoxicity with BCMA directed therapy.

B-cell maturation antigen (BCMA) has become a key target for antibody-drug conjugates, bispecific antibodies, chimeric antigen receptor T-cell therapies, and other immunotherapies in multiple myeloma. Some of these agents such as belantamab mafodotin and idecabtagene vicleucel have already received regulatory approval in the United States. Although BCMA has generally been considered to be expressed almost exclusively in plasma cells with a low likelihood of on-target off-tumor toxicity, there has been a range of unusual neurotoxicity observed across the spectrum of BCMA immunotherapies. In certain cases, these unusual neurotoxicity presentations have led to patient death or withdrawal of agents from further development. Our review summarizes the literature in this field and highlights the possibility of on-target toxicities due to neural expression of BCMA. We draw attention to the need for further investigation of these toxicities. This risk becomes increasingly important as BCMA targeted therapies are brought to earlier lines of treatment.

NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity.

CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies.See related commentary by Velasquez and Gottschalk, p. 559. This article is highlighted in the In This Issue feature, p. 549.

An Oncolytic Adenovirus Armed with a Nanobody Against CD47 Reprograms Tumor Immune Microenvironment and Drives Durable Antitumor Immunity

Background: Oncolytic virotherapy utilizes natural or engineered viruses to kill tumors selectively, representing a promising cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses (OVs) elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in don't eat me signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As ideal gene delivery vectors in cancer therapy, OVs can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Importantly, OVs can intratumorally deliver anti-CD47 antibodies without causing uncontrolled off-tumor toxicity. Methods: We developed a recombinant oncolytic adenovirus loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. An oncolytic adenovirus without transgene of interest was used as control. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer mouse models were established to evaluated in vivo antitumor efficacy of oncolytic adenoviruses. Findings: The recombinant oncolytic adenovirus armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice. Additionally, the recombinant oncolytic adenovirus elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in mice treated with the anti-CD47 oncolytic adenovirus; tumorigenesis was completely inhibited in these mice after tumor re-challenge. Interpretation: The recombinant anti-CD47 oncolytic adenovirus has an effectual antitumor activity and may be a promising antitumor agent. Funding Statement: This work was supported by the National Science and Technology Major Projects of New Drugs (2018ZX09201018-013), the National Science and Technology Major Project for Infectious Diseases Control (2017ZX10203206-004), the National Natural Science Foundation of China (81101728), Sichuan Regional Innovation Cooperation Project (20QYCX0100), the Innovation Spark Project of Sichuan University (2018SCUH0084). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, Chengdu, China.

Antibody-Drug Conjugates for the Treatment of Acute Pediatric Leukemia.

The clinical development of antibody–drug conjugates (ADCs) has gained momentum in recent years and these agents are gradually moving into frontline regimens for pediatric acute leukemias. ADCs consist of a monoclonal antibody attached to a cytotoxic payload by a cleavable linker. This structure allows for highly cytotoxic agents to be directly delivered to leukemia cells leading to cell death and avoids excessive off-tumor toxicity. Near universal expression on B-cell acute lymphoblastic leukemia (ALL) blasts and the ability of rapid internalization has rendered CD22 an ideal target for ADC in B-ALL. Inotuzumab ozogamicin, the anti-CD22 antibody linked to calicheamicin led to complete remission rates of 60–80% in patients with relapsed/refractory B-ALL. In acute myeloid leukemia (AML), the CD33 targeting gemtuzumab ozogamicin has demonstrated modest improvements in survival and is the only ADC currently licensed in the United States for pediatric patients with de novo AML. Several other ADCs have been developed and tested clinically for leukemia but have achieved limited success to date. The search for additional leukemia-specific targets and optimization of ADC structure and specificity are ongoing efforts to improve their therapeutic window. This review provides a comprehensive overview of ADCs in acute leukemias, with a focus on pediatric ALL and AML.