Mutation-associated neoantigens (MANAs) are highly cancer-specific targets for immunotherapy where peptides derived from intracellular mutant proteins are presented on the cell surface via HLA molecules. T cell-engaging bispecific antibodies and CAR T cells can target MANAs to eliminate cancer cells via T cell activation. However, the low antigen density of MANAs on the cell surface can limit therapeutic efficacy. Here, we investigated whether increasing the affinity of the H2 single-chain variable fragment (scFv) targeting the p53 R175H MANA (HMTEVVRHC presented on HLA-A*02:01) improves its therapeutic effect. We identified higher-affinity H2 variants via phage biopanning and a thiocyanate elution method. Increasing bispecific antibody affinity to the low nanomolar range increased cancer cell killing and tumor control in mouse xenograft models without sacrificing antigen specificity. We next asked how increasing scFv affinity impacts CAR T cell function - a matter of debate. We appended each variant scFv to a CD28z CAR, CD3γ, or the T cell receptor. In striking contrast to the bispecific antibody results, increasing CAR affinity decreased function in each CAR format due to lower T cell activation upon interaction with target cancer cells. These results have important implications for the design of future immunotherapeutic approaches targeting low-density antigens.

Structural biology has fundamentally influenced pharmaceutical research and development at Merck Sharp & Dohme LLC, Rahway, New Jersey, USA, progressing from pioneering macromolecular crystallography in the 1980s to a fully integrated platform that today encompasses X-ray crystallography, cryo-electron microscopy, micro-electron diffraction and cryo-electron tomography. In this review, we present a comprehensive overview of how these complementary methods have advanced drug discovery across diverse therapeutic areas. We illustrate how atomic to cellular structural insights inform drug discovery and development, from target identification and validation, hit finding, lead identification through lead optimization and clinical progression. Furthermore, we describe how structural biology techniques aid in formulation strategies of antibodies and vaccines. Finally, we highlight the growing integration of ex situ and in situ approaches as a paradigm shift towards elucidating drug mechanisms in native cellular contexts, a transition poised to accelerate the discovery and development of next-generation therapeutics.

Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.

The greatest challenge in cancer therapy is to eradicate cancer cells with minimal damage to normal cells. Targeted therapy has been developed to meet that challenge, showing a substantially increased therapeutic index compared with conventional cancer therapies. Antibodies are important members of the family of targeted therapeutic agents because of their extraordinarily high specificity to the target antigens. Therapeutic antibodies use a range of mechanisms that directly or indirectly kill the cancer cells. Early antibodies were developed to directly antagonize targets on cancer cells. This was followed by advancements in linker technologies that allowed the production of antibody-drug conjugates (ADCs) that guide cytotoxic payloads to the cancer cells. Improvement in our understanding of the biology of T cells led to the production of immune checkpoint-inhibiting antibodies that indirectly kill the cancer cells through activation of the T cells. Even more recently, bispecific antibodies were synthetically designed to redirect the T cells of a patient to kill the cancer cells. In this Review, we summarize the different approaches used by therapeutic antibodies to target cancer cells. We discuss their mechanisms of action, the structural basis for target specificity, clinical applications and the ongoing research to improve efficacy and reduce toxicity. In this Review, Paul et al. provide an overview of therapeutic antibodies as an important modality in cancer therapy today. They summarize the different approaches used by antibodies to target cancer cells including those of immune checkpoint inhibitors, bispecific antibodies and antibody-drug conjugates, as well as describing current strategies aimed at improving their efficacy and reducing toxicities.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the auto-regulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity.

NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.

Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRAS G12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.

Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [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 LB095.

The MID1 TRIM protein is important for ventral midline development in vertebrates, and mutations of its B-box1 domain result in several birth defects. The B-box1 domain of the human MID1 protein binds two zinc atoms and adopt a similar ββα-RING structure. This domain is required for the efficient ubiquitination of protein phosphatase 2A, alpha4, and fused kinase. Considering the structural similarity, the MID1 B-box1 domain exhibits mono-autoubiquitination activity, in contrast to poly-autoubiquitination observed for RING E3 ligases. To understand its mechanism of action, the interaction of the B-box1 domain with Ube2D1 (UbcH5a, E2), a preferred E2 ligase, is investigated. Using isothermal titration calorimetry, the MID1 RING and B-box1 domains were observed to have similar binding affinities with the Ube2D1 protein. However, NMR 15N-1H Heteronuclear Single Quantum Coherence titration, 15N relaxation data, and High Ambiguity Driven protein-protein DOCKing (HADDOCK) calculations show the B-box1 domain binding on a surface distinct from where RING domains bind. The novel binding interaction shows the B-box1 domain partially overlapping the noncovalent Ube2D1 and a ubiquitin binding site that is necessary for poly-autoubiquitination activity. The B-box1 domain also displaces the ubiquitin from the Ube2D1 protein. These studies reveal a novel binding interaction between the zinc-binding ββα-fold B-box1 domain and the Ube2D enzyme family and that this difference in binding, compared to RING E3 ligases, provides a rationale for its auto-monoubiquitination E3 ligase activity.

Background Antibodies' exquisite specificity enables the development of new targeted immunotherapies for treatment in the fight against cancer. Antibodies can be engineered to target peptides derived from oncogenic proteins that are presented on the cell surface by human leukocyte antigens (pHLA).1, 2 Here, we describe the structural basis of recognition of a T cell receptor (TCR)-mimic antibody, termed V2, against the HLA-A*03:01-restricted KRASG12V epitope (VVVGAVGVGK), highlighting its specificity. Methods For structural studies, the KRASG12V-specific full-length antibody (V2-IgG) was expressed in mammalian cells to generate an optimal protein fold. Structure determination of the V2-IgG in complex with KRASG12V/HLA-A*03:01 was performed by single particle cryo-electron microscopy (cryoEM). Two-dimensional and three-dimensional classifications were performed to produce a final refined map at 3.1 Å resolution. To fully understand determinants of the specificity of the V2 antibody, we determined the X-ray crystal structure of the KRASWT/HLA-A*03:01 monomer (resolution 2.6 Å). Biophysical characterization included carrying out differential scanning fluorimetry (DSF) and binding kinetics experiments using surface plasmon resonance (SPR). Furthermore, we screened a single variant library of the V2 TCRm antibody to identify key residues for peptide specificity and affinity enhancement. Results The cryoEM complex structure revealed the V2 TCRm antibody sits on top of the KRASG12V-HLA-A*03:01 binding groove, making multiple contacts and leaning heavily towards the C-terminus of the KRASG12V peptide. All contacts made between the V2 TCRm antibody and KRASG12V peptide were aliphatic and hydrophobic in nature, with no hydrogen bonds made directly with the peptide. Three complementarity determining regions (CDRs) of the V2 TCRm antibody formed a loose cage-like configuration around the G12V neoantigen. Structural alignment of the V2 bound KRASG12V-HLA-A*03:01 and unbound KRASWT-HLA-A*03:01 structures revealed the KRASG12V peptide underwent a conformational change upon antibody binding. This observation is in congruence with the SPR data that showed a two-state binding model. Moreover, the affinity matured process yielded V2 variant antibodies with comparable or enhanced affinity, but none retained both specificity and increased T-cell activation. Conclusions This complex structure is the first cryoEM structure of an antibody fragment binding a neoantigen-HLA target and the first structures of the KRASWT/G12V peptides presented by HLA-A*03:01 with or without an antibody in complex. Characterizing how the V2 TCRm antibody recognizes KRASG12V-HLA-A*03:01 and differences in KRASWT and KRASG12V peptide binding to HLA-A*03:01 offers insight into how highly hydrophobic peptide neoantigens can be targeted with antibody-based therapies. References Hsiue EH, Wright KM, Douglass J, Hwang MS, Mog BJ, Pearlman AH, Paul S, DiNapoli SR, Konig MF, Wang Q, Schaefer A, Miller MS, Skora AD, Azurmendi PA, Murphy MB, Liu Q, Watson E, Li Y, Pardoll DM, Bettegowda C, Papadopoulos N, Kinzler KW, Vogelstein B, Gabelli SB, Zhou S. Targeting a neoantigen derived from a common TP53 mutation. Science. 2021;371:eabc8697. Douglass J, Hsiue EH, Mog BJ, Hwang MS, DiNapoli SR, Pearlman AH, Miller MS, Wright KM, Azurmendi PA, Wang Q, Paul S, Schaefer A, Skora AD, Molin MD, Konig MF, Liu Q, Watson E, Li Y, Murphy MB, Pardoll DM, Bettegowda C, Papadopoulos N, Gabelli SB, Kinzler KW, Vogelstein B & Zhou S. Bispecific antibodies targeting mutant RAS neoantigens. Sci Immunol. 2021;6:eabd5515.

Voltage-gated sodium channels, Na V s, are responsible for the rapid rise of action potentials in excitable tissues. Na V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-af fi nity anti-Na V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle) channel isoforms. These Nbs were raised llama a phage display library high af fi nity the C-terminal (CT) of Nbs Escherichia , puri fi ed, high-af fi nity Nbs a Na V isoform they undesired cross-reactivity for different Na V isoforms. Nb17 Nb82 CTNa V 1.4 1.5 CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 Nb82 the CTNa V 1.4 and CTNa V 1.5 with high af fi nity ( K D (cid:1)

Voltage-gated sodium channels, Na(V)s, are responsible for the rapid rise of action potentials in excitable tissues. Na-V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na-V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na(V)1.4 (skeletal muscle) and Na(V)1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na(V)1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different Na-V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa(V)1.4 or CTNa(V)1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa(V)1.4 and CTNa(V)1.5 with high affinity (K-D similar to 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect Na(V)1.4 and Na(V)1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na(V)1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na-V reagents to capture Na(V)s from cell lysates and as molecular visualization agents for Na(V)s.

Chimeric antigen receptor (CAR) T cells have emerged as a promising class of therapeutic agents, generating remarkable responses in the clinic for a subset of human cancers. One major challenge precluding the wider implementation of CAR therapy is the paucity of tumor-specific antigens. Here, we describe the development of a CAR targeting the tumor-specific isocitrate dehydrogenase 2 (IDH2) with R140Q mutation presented on the cell surface in complex with a common human leukocyte antigen allele, HLA-B*07:02. Engineering of the hinge domain of the CAR, as well as crystal structure-guided optimization of the IDH2R140Q-HLA-B*07:02-targeting moiety, enhances the sensitivity and specificity of CARs to enable targeting of this HLA-restricted neoantigen. This approach thus holds promise for the development and optimization of immunotherapies specific to other cancer driver mutations that are difficult to target by conventional means.

Turning a tumor suppressor into a target Tumor-suppressor genes such as TP53 (tumor protein P53) play key roles in the pathogenesis of cancer but, unfortunately, they are difficult to target because they do not create an overactive protein that can be inhibited with a drug. Hsiue et al. discovered a way to target a cancer-associated mutant form of the p53 protein using the body's own immune system (see the Perspective by Weidanz). The authors identified a distinct fragment of this mutant protein, characterized the structural basis for its presentation to T cells, and designed a bispecific antibody to stimulate T cell killing of p53-mutant cancer cells. Science , this issue p. eabc8697 ; see also p. 996

Immunotherapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies redirect healthy T cells to kill cancer cells expressing the target antigen. The pan-B cell antigen-targeting immunotherapies have been remarkably successful in treating B cell malignancies. Such therapies also result in the near-complete loss of healthy B cells, but this depletion is well tolerated by patients. Although analogous targeting of pan-T cell markers could, in theory, help control T cell cancers, the concomitant healthy T cell depletion would result in severe and unacceptable immunosuppression. Thus, therapies directed against T cell cancers require more selective targeting. Here, we describe an approach to target T cell cancers through T cell receptor (TCR) antigens. Each T cell, normal or malignant, expresses a unique TCR β chain generated from 1 of 30 TCR β chain variable gene families (TRBV1 to TRBV30). We hypothesized that bispecific antibodies targeting a single TRBV family member expressed in malignant T cells could promote killing of these cancer cells, while preserving healthy T cells that express any of the other 29 possible TRBV family members. We addressed this hypothesis by demonstrating that bispecific antibodies targeting TRBV5-5 (α-V5) or TRBV12 (α-V12) specifically lyse relevant malignant T cell lines and patient-derived T cell leukemias in vitro. Treatment with these antibodies also resulted in major tumor regressions in mouse models of human T cell cancers. This approach provides an off-the-shelf, T cell cancer selective targeting approach that preserves enough healthy T cells to maintain cellular immunity.

Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here, we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt's kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution-phase assays and cell transfection experiments.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Sandra Gabelli1, Katharine Wright2, Micheller Miller3, Emily Han-Chung Hsiue4, Michael S. Hwang5, Puchong Thirawatananond6, P. Aitana Azurmendi7, Jackie Douglass8, Tihitina Aytenfisu9, Michael Murphy10, Brian J Mog11, Alexander H. Pearlman12, Drew M. Pardoll13 1Johns Hopkins University 2No affiliation given, 3Johns Hopkins University, 4Sidney Kimmel Comprehensive Cancer Center, 5Sidney Kimmel Comprehensive Cancer Center, 6Johns Hopkins University, 7Department of Biophysics and Biophysical Chemistry, 8Johns Hopkins University, 9Johns Hopkins University School of Medicine, 10Cytiva, 11Sidney Kimmel Comprehensive Cancer Center, 12Sidney Kimmel Comprehensive Cancer Center 13Sidney Kimmel Comprehensive Cancer Center gabelli@jhmi.edu

Background TP53 is the most commonly mutated cancer driver gene but drugs that target TP53 are not yet available. A peptide derived from the most common p53 mutation R175H (HMTEVVRHC) can be presented by a common human leukocyte antigen (HLA-A*02:01) after proteasomal degradation.1 We aimed to develop T cell receptor (TCR)-mimic antibody targeting this shared neoantigen. Methods We constructed a single-chain variable fragment (scFv) phage display library that presents scFvs at an estimated diversity of 3.6e10. Mutant peptide-HLA (pHLA)-specific scFvs were enriched through five rounds of positive and negative selections. Mutant pHLA-specific scFv clones were converted into bispecific T cell retargeting antibodies in the single-chain diabody (scDb) format by linking with the anti-CD3 scFv UCHT1.2 These scDbs were tested in T cell co-culturs in the presence of target pulsed cells or target cells that either overexpress the p53 neoantigen or present the p53 neoantigen at endogenous levels. In vivo efficacy was assessed by administering scDb in NSG xenograft mouse model. The structural basis of the binding specificity was evaluated by X-ray crystallography. Results We identified an scFv, termed clone H2, that specifically binds p53 R175H/HLA-A*02:01 pHLA and not its wild-type counterpart at a Kd of 86 nM (figure 1A). H2-scDb induced T cell cytokine release and cytotoxicity in the presence of 1) HLA-A*02:01-expressing cells pulsed with the p53R175H peptide, 2) cells transfected with HLA-A*02:01 and p53 R175H, and 3) cancer cell lines KMS26, KLE, and TYK-nu that express endogenous HLA-A*02:01/p53 R175H (figure 1B-E). T cell activation was diminished when TP53 was knocked out from these cancer cell lines using CRISPR (figure 1E). When administered to NOD scid gamma (NSG) mice systemically engrafted with the KMS-26 cell line, H2-scDb significantly suppressed tumor growth (figure 1 F, G). The structure of p53 R175H/HLA-A2 bound to the H2-Fab fragment shows that four complementarity-determining region loops of the H2 antibody formed a cage-like configuration around the C-terminus of the p53 R175H peptide, trapping the mutant histidine (His175) and the adjacent arginine (Arg174) residues in a stable interaction, which provides the structural basis for the specificity (figure 2). Conclusions We have developed a TCR-mimic bispecific T cell engager H2-scDb that recognized the shared neoantigen HLA-A*02:01/p53 R175H pHLA complex with exquisite specificity. It effectively activated T cells and lysed tumor cells both in vitro and in vivo. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways. Acknowledgements We thank Surojit Sur, Nicolas Wyhs, Ashley Cook, Marco Dal Molin, Richard L. Blosser, Lilian Dasko-Vincent, Christopher Thoburn, Jianhong Cao, and José Rodríguez Molina for assistance with this study. Trial Registration NA Ethics Approval NA Consent NA References Lo W, Parkhurst M, Robbins PF, Tran E, Lu YC, Jia L, Gartner JJ, Pasetto A, Deniger D, Malekzadeh P, Shelton TE, Prickett T, Ray S, Kivitz S, Paria BC, Kriley I, Schrump DS, Rosenberg SA. Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer. Cancer Immunol. Res 2019;7:534–543. Stork R, Campigna E, Robert B, Muller D, Kontermann RE. Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives. J Biol Chem 2009;284:25612–25619