Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.

Binding affinity is one of the primary determinants of antibody function. Here, we provide a protocol for simple and rapid affinity maturation of single-domain antibodies (sdAbs) using tandem phage display selection and next-generation DNA sequencing. The sequence of a model camelid sdAb directed against Clostridioides difficile toxin A (A26.8) was diversified using either random or site-saturation mutagenesis and cloned into a phagemid vector upstream of gene 3. The resulting phage-displayed sdAb libraries were panned against C. difficile toxin A and the panning outputs interrogated using Illumina MiSeq sequencing. Through bioinformatic analyses, we were able to identify individual affinity-enhancing amino acid substitutions in the sdAb complementarity-determining regions that, when combined, resulted in affinity improvements of approximately 10-fold. The advantages of this method are that it does not require extensive screening and characterization of individual clones, nor structural information on the mechanism of the sdAb:antigen interaction.

Biparatopic single-domain antibodies against Axl achieve ultra-high affinity through intramolecular engagement.

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHHs. The VHHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of VHHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl+ tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl+ cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These VHHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.

Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC.

Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: ∼6 µM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.

Serum albumin-binding VH Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75) demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

Single-Domain Antibodies Represent Novel Alternatives to Monoclonal Antibodies as Targeting Agents against the Human Papillomavirus 16 E6 Protein.

Approximately one fifth of all malignancies worldwide are etiologically associated with a persistent viral or bacterial infection. Thus, there is a particular interest in therapeutic molecules which use components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. Challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. Here, we addressed these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through construction and panning of two llama, immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these with recombinant E6 were further characterized using an enzyme-linked immunosorbent assay (ELISA), Western blotting under denaturing and native conditions, and surface plasmon resonance. Three VHHs were identified that bound recombinant E6 with nanomolar affinities. Our results lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.

Evaluation of a noncanonical Cys40-Cys55 disulfide linkage for stabilization of single-domain antibodies.

Incorporation of noncanonical disulfide linkages into single-domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T m s), reversibility of thermal unfolding, solubility, and antigen-binding affinities of three types of sdAbs (VH H, VH , and VL domains). The Cys40-Cys55 disulfide linkage was tolerated by 9/9 VH Hs, 12/12 VH s, and 2/11 VL s tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40-Cys55 disulfide linkage increased sdAb T m by an average of 10.0°C (range: 0-21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen-binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.

Isolation and characterization of camelid single-domain antibodies against HER2.

OBJECTIVE: To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2. RESULTS: We immunized a llama with human HER2, constructed a phage-displayed VHH library from the lymphocytes of the animal, and isolated six unique HER2-specific VHHs by panning. All six VHHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six VHHs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2+ SKOV3 cells by flow cytometry. Three of the VHHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The VHHs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These VHHs may be useful in the design of modular cancer immunotherapeutics.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display.

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.