CD4 Binding Research Articles

Abstract CT052: A phase 1/2 randomized study of imvotamab monotherapy and in combination with loncastuximab tesirine in relapsed/refractory non-Hodgkin lymphomas

Abstract Background: Recent advances in the treatment of non-Hodgkin lymphoma (NHL) have transformed the landscape and provided significant benefits to patients. Novel agents that target CD19 and CD20 on B cells, such as bispecific T-cell engagers and chimeric antigen receptor T-cell (CAR-T) therapy, have demonstrated benefit for patients in the relapsed and refractory setting. However, these agents can often be associated with significant toxicity and there is a need for new therapies that can provide clinical benefit with a superior safety profile. Imvotamab is a novel CD20 x CD3 bispecific antibody utilizing an IgM backbone. This allows targeting of up to 10 CD20 binding sites for every CD3 site. In preclinical models, imvotamab demonstrated encouraging antitumor activity and stimulated T-cells in a more physiologic manner than IgG-based antibodies, which may reduce adverse events typically associated with T-cell engagers and CAR-T, such as CRS and neurotoxicity. Loncastuximab tesirine is a CD19-targeting antibody-drug conjugate approved by the US FDA and EMA for relapsed DLBCL after 2 lines of systemic therapy. Loncastuximab tesirine has shown activity in both DLBCL and FL. Combining therapies such as loncastuximab tesirine, which targets CD19 and induce apoptosis of cancer cells, with imvotamab’s ability to eliminate CD20+ tumor cells by engagement with T-cells, may improve treatment outcomes among patients with NHL via synergistic mechanisms of action. Methods: This study is a Phase 1/2, multicenter, single-arm clinical trial of imvotamab as monotherapy and in combination with loncastuximab tesirine for patients with relapsed/refractory NHL. Phase 1a Dose Escalation is complete with no DLTs or neurotoxicity AEs up to 1000mg dose titration. Phase 1b Combination will evaluate imvotamab and loncastuximab tesirine in patients with R/R 2nd line or later NHL. Phase 2 monotherapy Dose Selection is currently ongoing. The Phase 2 component randomizes patients at two different dose levels (100 mg and 300 mg plateau dose) in two separate indications (R/R DLBCL and R/R FL). Patients receive weekly dosing on Day 1, 8, and 15 of each 21- day cycle. Dosing begins at 15 mg and is increased weekly for 3-4 weeks to reach the plateau dose. Patients then stay at the plateau dose until disease progression or unacceptable toxicity. Patients who achieve a response by Week 12 may switch to a less frequent dosing interval of every 3 weeks. Primary endpoints include frequency and severity of adverse events and objective response rate (ORR) based on Lugano criteria. Correlative biomarker studies will evaluate the relationship of clinical benefit with blood and tissue biomarkers. The study is currently open with patients enrolling in Phase 2 at time of submission. Phase 1b Combination is expected to begin enrollment in the first quarter of 2023. Clinical trial information: NCT04082936. Citation Format: Catherine Diefenbach, Won Seog Kim, Chan Cheah, Ajay K. Gopal, Philippe Armand, Ian Flinn, Gareth P. Gregory, Sung-Soo Yoon, Loretta Nastoupil, Jennifer Lue, Vincent Ribrag, Javier Briones, Antonio Salar-Silvestre, Anna Sureda-Balari, Matthew Ku, Hans Cruz, Paul Thaler, Ibrahim Qazi, Rachel Wei, Maya Leabman, Genevive Hernandez, Iris Sison, Elizabeth Budde. A phase 1/2 randomized study of imvotamab monotherapy and in combination with loncastuximab tesirine in relapsed/refractory non-Hodgkin lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT052.

Single-component multilayered self-assembling protein nanoparticles presenting glycan-trimmed uncleaved prefusion optimized envelope trimers as HIV-1 vaccine candidates

Uncleaved prefusion-optimized (UFO) design can stabilize diverse HIV-1 envelope glycoproteins (Envs). Single-component, self-assembling protein nanoparticles (1c-SApNP) can display 8 or 20 native-like Env trimers as vaccine candidates. We characterize the biophysical, structural, and antigenic properties of 1c-SApNPs that present the BG505 UFO trimer with wildtype and modified glycans. For 1c-SApNPs, glycan trimming improves recognition of the CD4 binding site without affecting broadly neutralizing antibodies (bNAbs) to major glycan epitopes. In mice, rabbits, and nonhuman primates, glycan trimming increases the frequency of vaccine responders (FVR) and steers antibody responses away from immunodominant glycan holes and glycan patches. The mechanism of vaccine-induced immunity is examined in mice. Compared with the UFO trimer, the multilayered E2p and I3-01v9 1c-SApNPs show 420 times longer retention in lymph node follicles, 20-32 times greater presentation on follicular dendritic cell dendrites, and up-to-4 times stronger germinal center reactions. These findings can inform future HIV-1 vaccine development.

Abstract 6345: Non-clinical pharmacology of HX009, a novel FIC PD1xCD47 BsAb

Abstract Immune checkpoint inhibitors (ICIs) have been widely tested as cancer therapeutics but with respective limitations such as marginal activity and/or poor safety profiles. It is possible that some of these limitations could be overcome by specially engineered molecules, e.g. bi-functional agents. Here we report a novel rationally designed bi-specific antibody (BsAb) targeting both PD1 and CD47, HX009, in order to improve both efficacy and safety over the two respective single-targeting ICIs. HX009 features IgG4 and 2 × 2 symmetric format, but with specifically weakened CD47 binding affinity. We performed a comprehensive preclinical pharmacology characterization of HX009 to test our hypothesis by confirming its candidacy as a novel first-in-class biologic for cancer therapy. The affinity constant (KD) of the HX009 binding to PD1 was determined to 3.96 × 10−11 M, while 5.06 × 10−9 M for binding to CD47, significantly reduced affinity as compared to the binding to PD1 and the binding between native SIRPα and CD47. HX009 has little binding to red blood cell (RBC) and platelets so to minimize hematological toxicity that usually associated with CD47 targeting. The antibody injectable formulation caused little cytokine release from PBMC (CRS or cytokine storm). HX009 cross-species binds to cynomolgus monkey PD1 and CD47 receptors, with the similar affinity as with the counterparts of human, but not to those of rodents. Cynomolgus monkey was thus chosen to investigate non-clinical pharmacokinetic (PK) and toxicology profiles of HX009. The intravenous (IV) infusion PK study revealed that HX009 is largely distributed within vasculature, with terminal half-life (T1/2) around 50hr and dose proportional exposure. Repeat dosing caused no accumulation. The production of antidrug antibody (ADA) were observed in the dosed animals, as expected for administration into different species, which impacted PK parameters of repeated dosing. The IV single dose toxicology study revealed the maximal tolerated dose (MTD) of 150mg/kg with 14-day observation, while the maximum non-severely toxic dose (HNSTD) was determined to be 15 mg/kg in the repeated dose study after once weekly dose for 4 consecutive weeks (5 times total). The main drug related toxicities observed seemed to be related to CD47 targeting as expected. Strong antitumor activity was also confirmed in in several preclinical cancer models, including humanized syngeneic models. In summary, the desired PK and safety profiles of HX009, along with anti-tumor activity, supporting further clinical development. Currently, HX009 is under clinical investigation (ClinicalTrials.gov Identifier: NCT05189093). Citation Format: Faming Zhang, Hang Ke, Feiyu Peng, Cen Chen, Lei Zhang, Henry Qixiang Li. Non-clinical pharmacology of HX009, a novel FIC PD1xCD47 BsAb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6345.

Abstract 855: Model-informed drug design (MIDD) exploration of a theoretical CD47xTAA bispecific antibody using available data from magrolimab, an anti-CD47 monoclonal antibody

Abstract Purpose: The goal of this study is to outline the process and utility of using fit-for-purpose, semi-mechanistic PK/PD modeling to inform the design of a theoretical CD47 bispecific antibody targeted to a generic tumor-associated antigen (CD47xTAA bsAb) with improved efficacy, tolerability, and PK compared to single target anti-CD47 therapies. Approach: To help guide the CD47xTAA bsAb design, a 3-compartment human model was built to include a target-mediated drug disposition (TMDD) component from CD47 expressed on red blood cells (RBC), white blood cells (WBC), and cells in the peripheral compartment. The model was parameterized using available literature data and calibrated against magrolimab human PK data that exhibited potential TMDD. The magrolimab model was used to identify key CD47 receptor occupancy (RO) thresholds associated with tolerability (the priming dose threshold) and efficacy (the recommended Phase 2 dose/RP2D threshold). Next, assumed drug properties and TAA expression levels for a CD47xTAA bsAb were implemented in a similar human model and drug PK and doses required to match key CD47 RO thresholds were explored across a range of single-arm binding affinities. Results: The calibrated magrolimab model projected target engagement levels on RBCs and WBCs and showed rapid antibody-induced CD47 pruning on RBCs that is consistent with clinical PD measurements. At TAA binding affinities that produce sufficient binding to tumor antigen, the model predicts that lowering the CD47 binding affinity allows for a higher starting dose that matches the priming threshold, while lowering the dose required to match the RP2D threshold. The model also shows that lowering the binding affinity to CD47 may improve PK exposure in the tumor compartment by reducing systemic CD47 TMDD. Conclusions: This modeling exercise demonstrates how semi-mechanistic PK/PD models can be used in model-informed drug design (MIDD) by leveraging the targeting advantage of avid bispecific antibody therapies and by using publicly available preclinical and clinical data. Furthermore, these model projections can help inform future studies with bispecific CD47 drug candidates. Citation Format: Emily A. Pace, Bjorn L. Millard. Model-informed drug design (MIDD) exploration of a theoretical CD47xTAA bispecific antibody using available data from magrolimab, an anti-CD47 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 855.

Abstract 1875: A novel T-cell-engaging therapeutical antibody against GPRC5D&BCMA for multiple myeloma treatment

Abstract T-cell-engaging therapeutical antibodies, such as Talquetamab (targeting GPRC5D and CD3 with 1:1 formation) and CC-93269(targeting BCMA and CD3 with 2:1 formation) have shown promise in multiple myeloma (MM) treatment. However, MM usually had heterogeneous cell populations with independent expressions of BCMA and GPRC5D, so TCEs only targeting BCMA or GPRC5D are suspected to have limitation on anti-tumor effect. Development a TCE simultaneously binding BCMA and GPRC5D may extend its therapeutic efficacy. MBS314 is a humanized tri-specific antibody against GPRC5D, BCMA, and CD3 for r/r MM treatment. MBS314 has a weaker binding to CD3 (KD=181nM) than Talquetamab (KD=25.6 nM) and CC-93269 (KD=41.0 nM). Meanwhile,it has high affinities to GPRC5D (KD=3.5nM) and BCMA (KD=0.22nM), similar to Talquetamab (KD=4.21 nM, to GPRC5D) and CC-93269 (KD=1.0 nM, to BCMA). We developed a reporter assay to evaluate the function of TCEs: co-culture the tumor cells, such as H929, AMO1, or KMS-11, with Jurkat-NFAT-Luc reporter cells can induce luciferase expression, which can reflect the CD3 agonist signaling. MBS314 stimulated CD3 signaling when the reporter cells cultured along with any of the above MM cells, while Talquetamab failed to stimulate CD3 signaling when the reporter cells cultured along with KMS-11, which has low expression of GPRC5D, and CC-93269 was unable to stimulate CD3 signaling with AMO1 which has low expression of BCMA. The cytotoxicity assay also indicated MBS314 mediated T cell killing any of the above cells, while Talquetamab failed to kill KMS-11 and CC-93269 killed AMO-1 with low efficiency. In our in vivo xenograft MM murine models, MBS314 and Talquetamab induced regression of established MM.1S tumor in all of the mice (4/4), while CC-93269 induced regression in half of the mice (2/4) with dose at 0.5mg/kg injected twice a week. And MBS314 inhibited the growth of the MOLP8 tumor, while Talquetamab and CC-93269 failed to prevent MOLP8 tumor growth. Especially, our ex vivo cytotoxicity assay with bone marrow mononuclear cells (BM-MNCs) from 15 MM patients indicated that MBS314 more efficiently induced T-cell-mediated depletion of CD138+ MM cells than Talquetamab (mean(EC50)=0.06nM vs.1nM) and CC-93269 (mean(EC50)= 0.06nM vs.0.33nM). In addition, owning to the low affinity to CD3, MBS314 has no detectable toxicity in CD3 humanized mice. In summary, our data show MBS314 is a promising TCE for MM treatment with high efficacy and broad spectrum targeting both GPRC5D and BCMA, and also has excellent safety with low CD3 binding affinity. Citation Format: Feng Li, Guojin Wu, Hao Peng, Jiangmei Li, Yue Qi, Guangzhong Lin, Huifang Liu, Wenqi Hu, Jinying Ning. A novel T-cell-engaging therapeutical antibody against GPRC5D&BCMA for multiple myeloma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1875.

Abstract 2942: ZW171, a T cell-engaging, bispecific antibody for the treatment of mesothelin-expressing solid tumors

Abstract Mesothelin (MSLN) is a GPI-linked membrane glycoprotein that is overexpressed in many cancer indications for which there is a high unmet medical need including pancreatic, mesothelioma, and ovarian. While MSLN-targeting agents have shown early signs of clinical activity, therapies with improved safety and efficacy are still needed. T cell engager (TCE) therapies have exhibited clinical utility against hematological malignancies, but limited success against solid tumors due to dose-limiting toxicities associated with cytokine release syndrome (CRS) and on-target off-tumor effects. To improve the therapeutic intervention of MSLN-expressing tumors, we engineered a bispecific TCE, designed to have an improved therapeutic window by enhancing both safety and anti-tumor activity profiles. To avoid dose-limiting toxicities related to CRS observed with high-affinity anti-CD3 paratopes (e.g. based on OKT3 or SP34), we engineered an anti-CD3 paratope (ZW_CD3_1) with low affinity CD3 binding and, in the context of a bispecific TCE, potent redirected T cell cytotoxicity. Compared to a bispecific antibody containing the high CD3 affinity SP34 paratope, the ZW_CD3_1 anti-CD3 paratope showed reduced and less potent T cell binding, stimulated less cytokine release yet elicited equivalent T cell mediated killing of MSLN-expressing tumor cells. Using the bispecific AzymetricTM platform, the ZW_CD3_1 paratope was incorporated into a panel of bispecific antibodies with different geometries, formats, and anti-MSLN paratope affinities then screened for T cell mediated lysis of target cells and production of cytokines. Based on reiterative screening and engineering, we determined the MSLN paratope affinity and the 2+1 format, consisting of two anti-MSLN single chain variable fragments (scFvs) and one anti-CD3 fragment antigen binding (Fab) domain, as the lead candidate format. In vivo, the lead format also showed significantly greater anti-tumor activity than other 2+1 bispecific formats. Following additional engineering and screening a lead MSLN x CD3 2 +1 bispecific antibody, ZW171, was selected for development. We further characterized the mechanistic and anti-tumor activity of ZW171 and showed that incubation of human T cells and tumor cells with ZW171 led to potent preferential killing of MSLN-mid and -high target cells while sparing MSLN-low expressing target cells. ZW171 only stimulated T cells to proliferate and produce cytokines in the presence of MSLN-expressing tumor cells, mitigating the risk of peripheral T cell activation and CRS. Importantly, ZW171 exhibited potent tumor growth inhibition in multiple established tumor models. Collectively, these data suggest that ZW171 has the potential to be an efficacious and safe therapeutic for the treatment of MSLN-expressing cancers. Citation Format: Nicole J. Afacan, Chayne Piscitelli, Patricia Zwierzchowski, Siran Cao, Janessa Li, Wingkie Wong, Kara White-Moyes, Thomas Spreter von Kreudenstein, Nina E. Weisser. ZW171, a T cell-engaging, bispecific antibody for the treatment of mesothelin-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2942.

Abstract 2971: EVOLVETM: A novel T cell engager platform with integrated CD2 costimulation engineered for the treatment of immune suppressive tumors

Abstract Bispecific antibodies which bind a tumor antigen and the CD3 heterodimer of the T cell receptor to form a synthetic immunological synapse represent a class of T cell engagers that has been clinically validated with the approvals of blinatumomab (Blinctyo®) and mosunetuzumab (Lunsumio®), targeting CD19 and CD20 respectively, for the treatment of B cell acute lymphoblastic leukemia and follicular lymphoma. Despite being a potent class of therapeutics, not all patients respond to therapy. Recent studies into state of blinatumomab-treated T cells highlight exhaustion and the lack of co-stimulation as potential factors in resistance. In solid tumors, the immunosuppressive tumor microenvironment is also a major factor while cytokine release is a safety consideration for all bispecifics that bind CD3 with strong affinity. To address the challenges of CD3 bispecifics, we have developed the EVOLVE™ platform, a trispecific antibody that consists of tumor targeting and attenuated CD3 binding affinity coupled with a CD2 agonist. We show that CD2 costimulation is superior to other pathways in its ability to sustain T cell activation and expansion and cytokine production over repeated cycles of stimulation. By integrating a CD2 agonist with an attenuated CD3 affinity bispecific, we can restore cytolytic potential without concomitant increase in cytokine release as compared to a strong CD3 affinity bispecific. The EVOLVE platform is also capable of inducing tumor cell killing by in vitro exhausted T cells whereas the bispecific did not. In terms of safety, the EVOLVE platform does not induce peripheral T cell activation and cytokine release in PBMCs in the absence of target as compared to CD3 and CD28 agonist antibodies. The superiority of the EVOLVE platform compared to a bispecific was also demonstrated in vivo where we observed 8/8 complete responses in EVOLVE-treated animals whereas the strong CD3 affinity bispecific showed no difference compared to isotype control using a CORL105 lung cancer xenograft model. We believe these experiments recapitulate the continual clinical challenges of using strong CD3 affinity bispecifics while demonstrating the advantages of the EVOLVE platform. Finally, we demonstrate the modular nature of the EVOLVE platform across diverse tumor antigens including B7H4, PSMA, CD20, and a novel squamous tumor antigen ULBP2. Together our data highlight the broad applications of the EVOLVE platform to improve T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mohosin Sarkar, Eric M. Tam, Guixian Jin, Shu Shien Chin, Abudukadier Abulizi, Oksana A. Sergeeva, Zengzu Lai, Evelyn Teran, Hayden Karp, Danielle Klaskin, Nana Adjoa Pels, Xingyue An, Jennifer Ziegler, Changqing Yuan, Maria Hackettt, Sonali Dhindwal, Amber Fearnley, Julio Rodriguez, Louis Matis, Jay S. Fine, Jeremy S. Myers. EVOLVETM: A novel T cell engager platform with integrated CD2 costimulation engineered for the treatment of immune suppressive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2971.

HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models

Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009’s further clinical development for treating NHLs.

Hepatitis C virus E1 and modified E2 delivered from an mRNA vaccine induces protective immunity

Hepatitis C virus (HCV) is characterized by a high number of chronic cases due to an impairment of protective innate and adaptive immune responses. Here, we examined the contribution of the individual ectodomains of E1, E2, or a modified E2 with reduced CD81 binding and an inserted N-linked glycosylation site in combination as vaccine antigen mRNA-lipid nanoparticles (LNPs). The induction of a protective immune response to surrogate recombinant vaccinia virus (VV) expressing homologous HCV glycoprotein(s) challenge infection in a BALB/c mouse model was observed. Vaccination with a mRNA-LNP expressing soluble E1 (sE1) significantly reduced vv/HCV titer in the mouse ovary. However, the addition of sE2 mRNA-LNP for immunization impaired the efficacy of the sE1 construct. Further analysis showed that Th1 related cytokine responses to the sE1 mRNA-LNP were significantly altered in the presence of sE2 following co-immunization. Evaluation of immunogenicity revealed that the use of modified sE2F442NYT nucleoside mRNA-LNP vaccine results in an improved cellular immune response, IgG2a isotype switching, enhanced total IgG, and an increase in the neutralizing antibody response against HCV pseudotype virus. HCV cross genotype specific reactivity to peptides representing conserved E2 specific linear epitopes were enhanced in modified E2 vaccinated animal sera. In the absence of a suitable immunocompetent small animal model for HCV infection, protection from surrogate HCV vaccinia challenge infection model was observed in the immunized mice as compared to sE1 alone or an unmodified sE2 mRNA-LNP vaccine. Inclusion of sE1 with modified sE2F442NYT as mRNA-LNP vaccine candidate appeared to be beneficial for protection.

Naturally occurring dipeptide from elite controllers with dual anti-HIV-1 mechanism

BackgroundEnhanced levels of a dipeptide, WG-am, have been reported among elite controllers – patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WG-am. MethodsDrug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strainswere performed to evaluate the antiviral mechanism of WG-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WG-am. ResultsThe data suggest that WG-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WG-am also inhibited HIV-1 at 4–6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WG-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WG-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WG-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). ConclusionNaturally occurring in HIV-1 elite controllers, WG-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WG-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WG-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.

Nanobody‐mediated complement activation to kill HIV‐infected cells

The complement system which is part of the innate immune response against invading pathogens represents a powerful mechanism for killing of infected cells. Utilizing direct complement recruitment for complement‐mediated elimination of HIV‐1‐infected cells is underexplored. We developed a novel therapeutic modality to direct complement activity to the surface of HIV‐1‐infected cells. This bispecific complement engager (BiCE) is comprised of a nanobody recruiting the complement‐initiating protein C1q, and single‐chain variable fragments of broadly neutralizing antibodies (bNAbs) targeting the HIV‐1 envelope (Env) protein. Here, we show that two anti‐HIV BiCEs targeting the V3 loop and the CD4 binding site, respectively, increase C3 deposition and mediate complement‐dependent cytotoxicity (CDC) of HIV‐1 Env‐expressing Raji cells. Furthermore, anti‐HIV BiCEs trigger complement activation on primary CD4 T cells infected with laboratory‐adapted HIV‐1 strain and facilitates elimination of HIV‐1‐infected cells over time. In summary, we present a novel approach to direct complement deposition to the surface of HIV‐1‐infected cells leading to complement‐mediated killing of these cells.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3.As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

Structural basis of antibodies targeting neutralizing epitopes revealed by cryoempem

Engineered HIV-1 envelope glycoprotein immunogens can be designed to activate naïve B cells expressing germline antibody precursors that have the potential to evolve into broadly neutralizing antibodies. We applied cryo-EM based polyclonal epitope mapping (cryoEMPEM) to characterize the polyclonal antibody responses elicited by rhesus macaques immunized with a germline-targeting stabilized Env (BG505 SOSIP GT1.1). Antibodies against two neutralizing epitopes, CD4 binding site (CD4bs) and fusion peptide, were identified along with some non-neutralizing or strain-specific epitopes (V5 loop, glycans holes and base). A hybrid structural and bioinformatic approach was applied to predict monoclonal antibody sequences from the cryoEMPEM maps. Using IgDiscover and next-generation sequencing results, we identified candidate variable region sequences compatible with the amino acid side chain densities of CD4bs- or FP-directed cryoEMPEM responses. These monoclonal antibodies were then expressed and purified to confirm their binding to BG505 SOSIP GT1.1 antigens by biolayer interferometry and EM. Superimposition of the monoclonal antibody cryoEM models with the corresponding polyclonal cryoEMPEM model revealed high similarity, further demonstrating the efficacy of our structure-to-sequence method for antibody discovery. Overall, our work reveals that cryoEMPEM-identified antibodies targeting intended neutralizing epitopes were induced by BG505 SOSIP GT1.1 trimers in rhesus macaques and we successfully inferred monoclonal sequences from the structural data. Our structure-to-sequence pipeline offers an alternative to typical monoclonal antibody isolation strategies and is a promising complement to the future of iterative vaccine design.

Acute Inflammatory Recovery-A Matter of Sophisticated Interaction of Two Cell Populations?

Acute Inflammatory Recovery-A Matter of Sophisticated Interaction of Two Cell Populations?

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function–Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants

T-cell engaging bispecific antibodies (TCBs) targeting CD3 and tumor-specific antigens are very promising therapeutic modalities. Since CD3 binding is crucial for the potency of TCBs, understanding the functional impact of CD3 antigen-binding fragment modifications is of utmost importance for defining critical quality attributes (CQA). The current CQA assessment strategy requires the integration of structure-based physicochemical separation and functional cell-based potency assays. However, this strategy is tedious, and coexisting proteoforms with potentially different functionalities may not be individually assessed. This increases the degree of ambiguities for defining meaningful CQAs, particularly for complex bispecific antibody formats such as TCBs. Here, we report for the first time a proof-of-concept study to separate and identify critically modified proteoforms of TCBs using functional CD3 target affinity chromatography (AC) coupled with online mass spectrometry (MS). Our method enabled functional distinction of relevant deamidated and glycosylated proteoforms and the simultaneous assessment of product-related variants such as TCB mispairings. For example, CD3 AC-MS allowed us to separate TCB mispairings with increased CD3 binding (i.e., knob-knob homodimers) within the bound fraction. The functional separation of proteoforms was validated using an established workflow for CQA identification based on thoroughly characterized ion-exchange fractions of a 2+1 TCB. In addition, the new method facilitated the criticality assessment of post-translational modifications in stress studies and structural variants in early stage clone selection. CD3 AC-MS has high impact for streamlining the integration of functional and structural characterizations of the large landscape of therapeutic CD3 targeting TCBs from early stage research to late stage characterization.

IMM0306, a fusion protein of CD20 mAb with the CD47 binding domain of SIRPα, exerts excellent cancer killing efficacy by activating both macrophages and NK cells via blockade of CD47-SIRPα interaction and FcɣR engagement by simultaneously binding to CD47 and CD20 of B cells

IMM0306, a fusion protein of CD20 mAb with the CD47 binding domain of SIRPα, exerts excellent cancer killing efficacy by activating both macrophages and NK cells via blockade of CD47-SIRPα interaction and FcɣR engagement by simultaneously binding to CD47 and CD20 of B cells

Characterization of a Novel CD4 Mimetic Compound YIR-821 against HIV-1 Clinical Isolates.

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821. IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required.

Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies.

Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified. In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes. In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro. Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

Structural and Functional Characterization of Indane-Core CD4-Mimetic Compounds Substituted with Heterocyclic Amines.

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer on the virion surface interacts with the host receptors, CD4 and CCR5/CXCR4, to mediate virus entry into the target cell. CD4-mimetic compounds (CD4mcs) bind the gp120 Env, block CD4 binding, and inactivate Env. Previous studies suggested that a C(5)-methylamino methyl moiety on a lead CD4mc, BNM-III-170, contributed to its antiviral potency. By replacing the C(5) chain with differentially substituted pyrrolidine, piperidine, and piperazine ring systems, guided by structural and computational analyses, we found that the 5-position of BNM-III-170 is remarkably tolerant of a variety of ring sizes and substitutions, both in regard to antiviral activity and sensitization to humoral responses. Crystallographic analyses of representative analogues from the pyrrolidine series revealed the potential for 5-substituents to hydrogen bond with gp120 Env residue Thr 283. Further optimization of these interactions holds promise for the development of CD4mcs with greater potency.

Abstract B04: Preclinical activity and safety profile or JANX008, a novel EGFR-targeting tumor-activated T cell engager for treatment of solid tumors

Abstract Introduction: Epidermal growth receptor (EGFR) is the most expressed membrane oncogenic protein in human cancers. KRAS and BRAF mutations are significant drivers of resistance to EGFR-targeted therapies. Unlike other treatments, EGFR-targeting, CD3 bispecific T cell engagers (TCEs) can potentially retain activity against tumors bearing resistance mutations. However, cytokine release syndrome (CRS), on-target off-tumor toxicities, and poor pharmacokinetics (PK) properties present significant clinical limitations for these potent immunomodulators. To overcome these challenges, Janux has developed JANX008, an EGFR- and CD3-targeted tumor-activated T cell engager (TRACTr). JANX008 is a humanized tri-specific protein that contains EGFR- and CD3-binding domains, an albumin binding domain to extend circulating half-life, and two different peptide masks fused to the molecule through tumor protease cleavable linkers. One peptide mask inhibits EGFR engagement on target cells, and the other inhibits CD3 engagement on T cells. Once the cleavage sequences undergo proteolysis by tumor proteases, the EGFR and CD3 masks are released, and the resulting active molecule can bind EGFR and CD3 on target cells. Methods: Peptide masks against EGFR- and CD3-binding domains were identified via phage display. The efficiency of the masks was evaluated using human EGFR and CD3 ELISAs. JANX008-induced cleavage-dependent T cell killing was evaluated in human PBMC/tumor cell co-culture assays. Anti-tumor efficacy of JANX008 was tested in multiple preclinical models, including EGFR antibody-resistant KRAS- and PIK3CA-mutant mouse colon cancer model (HCT116) and a fully human primary colorectal cancer (CRC) tumoroid system. The pharmacokinetic and safety profile of JANX008 was evaluated in non-human primate studies. Results: JANX008 target engagement was cleavage-dependent where masking reduced EGFR and CD3 binding by >300x and >1,000x, respectively. JANX008 exhibited potent cleavage- and dose-dependent activity in multiple preclinical models, including EGFR antibody-resistant tumor and T cell co-culture assays, humanized mouse CRC model, and a human primary CRC tumoroids with an intact tumor microenvironment. JANX008 showed a significantly enhanced safety profile in NHPs compared to non-masked EGFR-TCE, including decreased CRS-associated cytokines and healthy tissue toxicities at high exposures. Clinical chemistry, hematology, and pathology measurements all supported No-Observed-Adverse-Effect-Level ≥ 0.6 mg/kg/dose. Finally, the cleavable albumin-binding domain extended the half-life of JANX008 to ~94h, relative to the ~1.3h half-life of non-masked TCE, supporting its weekly clinical dosing. Conclusions: Preclinical data demonstrate key characteristics of JANX008, including cleavage-dependent activity, half-life extended PK, the potential for superior safety, and manufacturability properties that could mitigate significant limitations of TCEs and support JANX008 clinical development. Citation Format: Thomas R DiRaimondo, Natalija Budimir, Lina Ma, Simon Shenhav, Vanessa Cicchini, Hu Wu, Renee Jocic, Fabrece Roup, Calvin Campbell, Carolina Caffaro, Hans Aerni, Ugur Eskiocak, Wayne Godfrey, Charles Winter, Marc Nasoff, Neil Gibson, David Campbell, shahram Salek-Ardakani. Preclinical activity and safety profile or JANX008, a novel EGFR-targeting tumor-activated T cell engager for treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B04.