Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Surface and Global Proteome Analyses Identify ENPP1 and Other Surface Proteins as Actionable Immunotherapeutic Targets in Ewing Sarcoma.

PURPOSE: Ewing sarcoma is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. Although the survival rate of patients diagnosed with localized disease is approximately 70%, this decreases to approximately 30% for patients with metastatic disease and only approximately 10% for treatment-refractory disease, which have not changed for decades. Therefore, new therapeutic strategies are urgently needed for metastatic and refractory Ewing sarcoma. EXPERIMENTAL DESIGN: This study analyzed 19 unique Ewing sarcoma patient- or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared with a reference standard of 12 immortalized non-Ewing sarcoma cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to complement the surfaceome data. All models were analyzed by Tandem Mass Tags-based mass spectrometry to quantify identified proteins. RESULTS: The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known Ewing sarcoma-associated proteins, including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in Ewing sarcoma tumors compared with other childhood sarcomas and normal tissues. CONCLUSIONS: Our comprehensive proteomic characterization of the Ewing sarcoma surfaceome provides a rich resource of surface-expressed proteins in Ewing sarcoma. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in Ewing sarcoma. See related commentary by Bailey, p. 934.

Assessment of Novel Mesothelin-Specific Human Antibody Domain VH-Fc Fusion Proteins-Based PET Agents.

Mesothelin (MSLN) is a tumor-associated antigen found in a variety of cancers and is a target for imaging and therapeutic applications in MSLN-expressing tumors. We have developed high affinity anti-MSLN human VH domain antibodies, providing alternative targeting vectors to conventional IgG antibodies that are associated with long-circulating half-lives and poor penetration of tumors, limiting antitumor activity in clinical trials. Based on two newly identified anti-MSLN VH binders (3C9, 2A10), we generated VH-Fc fusion proteins and modified them for zirconium-89 radiolabeling to create anti-MSLN VH-Fc PET tracers. The focus of this study was to assess the ability of PET-imaging to compare the in vivo performance of anti-MSLN VH-Fc fusion proteins (2A10, 3C9) targeting different epitopes of MSLN vs IgG1 (m912; a clinical benchmark antibody with an overlapped epitope as 2A10) for PET imaging in a mouse model of colorectal cancer (CRC). The anti-MSLN VH-Fc fusion proteins were successfully modified and radiolabeled with zirconium-89. The resulting MSLN-targeted PET-imaging agents demonstrated specific uptake in the MSLN-expressing HCT116 tumors. The in vivo performance of the MSLN-targeted PET-imaging agents utilizing VH-Fc showed more rapid and greater accumulation and deeper penetration within the tumor than the full-length IgG1 m912-based PET-imaging agent. Furthermore, PET imaging allowed us to compare the pharmacokinetics of epitope-specific VH domain-based PET tracers. Overall, these data are encouraging for the incorporation of PET imaging to assess modified VH domain structures to develop novel anti-MSLN VH domain-based therapeutics in MSLN-positive cancers as well as their companion PET imaging agents.

Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

Human antibody VH domains targeting uPAR as candidate therapeutics for cancers.

The high expression of uPAR has been linked to tumor progression, invasion, and metastasis in several types of cancer. Such overexpression of uPAR makes it a potential target for immunotherapies across common cancers such as breast, colorectal, lung, ovarian cancer, and melanoma. In our study, two high-affinity and specific human VH domain antibody candidates, designed as clones 3 and 115, were isolated from a phage-displayed human VH antibody library. Domain-based bispecific T- cell engagers (DbTE) based on these two antibodies exhibited potent killing of uPAR-positive cancer cells. Thus, these two anti-uPAR domain antibodies are promising candidates for treating uPAR positive cancers.

Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma.

Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate.

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3 nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8 h post-infusion and remained at the site for over 10 days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

Rapid Emergence of Potentially Transmissible Severe Acute Respiratory Syndrome Coronavirus 2 With Resistance to Combination Monoclonal Antibody Therapy.

Prolonged coronavirus disease 2019 may generate new viral variants. We report an immunocompromised patient treated with monoclonal antibodies who experienced rebound of viral RNA and emergence of an antibody-resistant (>1000-fold) variant containing 5 mutations in the spike gene. The mutant virus was isolated from respiratory secretions, suggesting the potential for secondary transmission.

Human Antibody VH Domains Targeting GPNMB and VCAM-1 as Candidate Therapeutics for Cancers.

The elevated expression of GPNMB and VCAM-1 has been observed in many cancers including breast cancer, melanoma, and prostate cancers. Such overexpression of GPNMB and VCAM-1 has been associated with poor prognosis and increased cancer metastasis. Thus, GPNMB and VCAM-1 are potential targets for immunotherapies across multiple cancers. In this study, two high-affinity specific human VH domain antibody candidates, 87 (GPNMB) and 1B2 (VCAM-1), were isolated from our in-house proprietary phage-displayed human VH antibody domain libraries. The avidity was increased after conversion to VH-Fc. Domain-based bispecific T-cell engagers (DbTE) based on these two antibodies combined with the anti-CD3ε OKT3 antibody exhibited potent killing against GPNMB and VCAM-1-positive cancer cells, respectively. Hence, these two domain antibodies are promising therapeutic candidates for cancers expressing GPNMB or VCAM-1.

Development of an efficient method for selection of stable cell pools for protein expression and surface display with Expi293F cells.

Compare with transient expression, stable cell lines generally have higher productivity and better quality for protein expression. However, selection of stable cell line is time-consuming and laborious. Here we describe an optimized selection method to achieve high-efficient stable cell pools with Expi293F suspension cells. This method only takes 2-3 weeks to generate stable cell pools with 2- to 10-fold higher productivity than transient gene expression (TGE). In fed-batch culture with Yeastolate, >1 g/L yield was achieved with our KTN0239-IgG stable cell pool in shaker flasks. This method can be also applied to efficiently display proteins on the cell surface.

COVID-19 convalescent plasma boosts early antibody titer and does not influence the adaptive immune response.

Multiple randomized, controlled clinical trials have yielded discordant results regarding the efficacy of convalescent plasma in outpatients, with some showing an approximately 2-fold reduction in risk and others showing no effect. We quantified binding and neutralizing antibody levels in 492 of the 511 participants from the Clinical Trial of COVID-19 Convalescent Plasma in Outpatients (C3PO) of a single unit of COVID-19 convalescent plasma (CCP) versus saline infusion. In a subset of 70 participants, peripheral blood mononuclear cells were obtained to define the evolution of B and T cell responses through day 30. Binding and neutralizing antibody responses were approximately 2-fold higher 1 hour after infusion in recipients of CCP compared with saline plus multivitamin, but levels achieved by the native immune system by day 15 were almost 10-fold higher than those seen immediately after CCP administration. Infusion of CCP did not block generation of the host antibody response or skew B or T cell phenotype or maturation. Activated CD4+ and CD8+ T cells were associated with more severe disease outcome. These data show that CCP leads to a measurable boost in anti-SARS-CoV-2 antibodies but that the boost is modest and may not be sufficient to alter disease course.

Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates.

Chimeric antigen receptor-T (CAR-T) cells and antibody-drug conjugates (ADCs) are promising therapeutic strategies in oncology. The carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is overexpressed in tumors including non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC), and is an attractive target for therapies based on CAR-T cell or/and ADCs. We previously developed a highly specific antibody-based CAR-T cells targeting CEACAM5 and the tumoricidal effect of CAR-T cells was proved against neuro-endocrine prostate cancer (NEPC) cells expressing CEACAM5. Here, we compare the anti-tumor efficacy of our CAR-T cells with that of an anti-CEACAM5 ADC being clinically evaluated against NSCLC. Our anti-CEACAM5 CAR-T cells showed cytotoxicity in a CEACAM5 surface concentration dependent manner and reduced tumor growth in both ADC-responsive and -non-responsive CEACAM5-expressing NSCLC cells in vitro and in vivo. In contrast, the ADC exhibited cytotoxicity independent on the CEACAM5 cell surface concentration. Even though clinical translation of CEACAM5 targeting CAR-T cell therapies is still in preclinical stage, our CAR-T cell approach could provide a potential therapeutic strategy for CEACAM5-positive cancer patients with resistance to ADCs.

Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells.

Metastasis and chemoresistance in colorectal cancer are mediated by certain poorly differentiated cancer cells, known as cancer stem cells, that are maintained by Notch downstream signaling initiated upon Notch cleavage by the metalloprotease ADAM10. It has been shown that ADAM10 overexpression correlates with aberrant signaling from Notch, erbBs, and other receptors, as well as a more aggressive metastatic phenotype, in a range of cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia. ADAM10 inhibition, therefore, stands out as an important and new approach to deter the progression of advanced CRC. For targeting the ADAM10 substrate-binding region, which is located outside of the catalytic domain of the protease, we generated a human anti-ADAM10 monoclonal antibody named 1H5. Structural and functional characterization of 1H5 reveals that it binds to the substrate-binding cysteine-rich domain and recognizes an activated ADAM10 conformation present on tumor cells. The mAb inhibits Notch cleavage and proliferation of colon cancer cell lines in vitro and in mouse models. Consistent with its binding to activated ADAM10, the mAb augments the catalytic activity of ADAM10 towards small peptide substrates in vitro. Most importantly, in a mouse model of colon cancer, when administered in combination with the therapeutic agent Irinotecan, 1H5 causes highly effective tumor growth inhibition without any discernible toxicity effects. Our singular approach to target the ADAM10 substrate-binding region with therapeutic antibodies could overcome the shortcomings of previous intervention strategies of targeting the protease active site with small molecule inhibitors that exhibit musculoskeletal toxicity.

Human antibodies targeting ENPP1 as candidate therapeutics for cancers.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein expressed in many tissues. High expression levels of ENPP1 have been observed in many cancer types such as lung cancer, ovarian cancer, and breast cancer. Such overexpression has been associated with poor prognosis in these diseases. Hence, ENPP1 is a potential target for immunotherapy across multiple cancers. Here, we isolated and characterized two high-affinity and specific anti-ENPP1 Fab antibody candidates, 17 and 3G12, from large phage-displayed human Fab libraries. After conversion to IgG1, the binding of both antibodies increased significantly due to avidity effects. Based on these antibodies, we generated antibody-drug conjugates (ADCs), IgG-based bispecific T-cell engagers (IbTEs), and CAR T-cells which all exhibited potent killing of ENPP1-expressing cells. Thus, these various antibody-derived modalities are promising therapeutic candidates for cancers expressing human ENPP1.

GPC2 antibody-drug conjugate reprograms the neuroblastoma immune milieu to enhance macrophage-driven therapies.

BACKGROUND: Antibody-drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote additional endogenous antitumor immune responses. Here, we characterized the immunomodulatory properties of D3-GPC2-PBD, a pyrrolobenzodiazepine (PBD) dimer-bearing ADC that targets glypican 2 (GPC2), a cell surface oncoprotein highly differentially expressed in neuroblastoma. METHODS: ADC-mediated induction of ICD was studied in GPC2-expressing murine neuroblastomas in vitro and in vivo. ADC reprogramming of the neuroblastoma tumor microenvironment was profiled by RNA sequencing, cytokine arrays, cytometry by time of flight and flow cytometry. ADC efficacy was tested in combination with macrophage-driven immunoregulators in neuroblastoma syngeneic allografts and human patient-derived xenografts. RESULTS: The D3-GPC2-PBD ADC induced biomarkers of ICD, including neuroblastoma cell membrane translocation of calreticulin and heat shock proteins (HSP70/90) and release of high-mobility group box 1 and ATP. Vaccination of immunocompetent mice with ADC-treated murine neuroblastoma cells promoted T cell-mediated immune responses that protected animals against tumor rechallenge. ADC treatment also reprogrammed the tumor immune microenvironment to a proinflammatory state in these syngeneic neuroblastoma models, with increased tumor trafficking of activated macrophages and T cells. In turn, macrophage or T-cell inhibition impaired ADC efficacy in vivo, which was alternatively enhanced by both CD40 agonist and CD47 antagonist antibodies. In human neuroblastomas, the D3-GPC2-PBD ADC also induced ICD and promoted tumor phagocytosis by macrophages, which was further enhanced when blocking CD47 signaling in vitro and in vivo. CONCLUSIONS: We elucidated the immunoregulatory properties of a GPC2-targeted ADC and showed robust efficacy of combination immunotherapies in diverse neuroblastoma preclinical models.

In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).