Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) is known to be critically involved in tissue development and homeostasis as well as in the pathogenesis of cancer. Here we showed that Foxp3(+) regulatory T (Treg) cells express EGFR under inflammatory conditions. Stimulation with the EGF-like growth factor Amphiregulin (AREG) markedly enhanced Treg cell function in vitro, and in a colitis and tumor vaccination model we showed that AREG was critical for efficient Treg cell function in vivo. In addition, mast cell-derived AREG fully restored optimal Treg cell function. These findings reveal EGFR as a component in the regulation of local immune responses and establish a link between mast cells and Treg cells. Targeting of this immune regulatory mechanism may contribute to the therapeutic successes of EGFR-targeting treatments in cancer patients.

Highly specific and potently activating Vγ9Vδ2-T cell specific nanobodies for diagnostic and therapeutic applications.

Vγ9Vδ2-T cells constitute the predominant subset of γδ-T cells in human peripheral blood and have been shown to play an important role in antimicrobial and antitumor immune responses. Several efforts have been initiated to exploit these cells for cancer immunotherapy, e.g. by using phosphoantigens, adoptive cell transfer, and by a bispecific monoclonal antibody based approach. Here, we report the generation of a novel set of Vγ9Vδ2-T cell specific VHH (or nanobody). VHH have several advantages compared to conventional antibodies related to their small size, stability, ease of generating multispecific molecules and low immunogenicity. With high specificity and affinity, the anti-Vγ9Vδ2-T cell receptor VHHs are shown to be useful for FACS, MACS and immunocytochemistry. In addition, some VHH were found to specifically activate Vγ9Vδ2-T cells. Besides being of possible immunotherapeutic value, these single domain antibodies will be of great value in the further study of this important immune effector cell subset.

Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

Therapeutic stem cells expressing variants of EGFR-specific nanobodies have antitumor effects.

The deregulation of the epidermal growth factor receptor (EGFR) has a significant role in the progression of tumors. Despite the development of a number of EGFR-targeting agents that can arrest tumor growth, their success in the clinic is limited in several tumor types, particularly in the highly malignant glioblastoma multiforme (GBM). In this study, we generated and characterized EGFR-specific nanobodies (ENb) and imageable and proapoptotic ENb immunoconjugates released from stem cells (SC) to ultimately develop a unique EGFR-targeted therapy for GBM. We show that ENbs released from SCs specifically localize to tumors, inhibit EGFR signaling resulting in reduced GBM growth and invasiveness in vitro and in vivo in both established and primary GBM cell lines. We also show that ENb primes GBM cells for proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Furthermore, SC-delivered immunoconjugates of ENb and TRAIL target a wide spectrum of GBM cell types with varying degrees of TRAIL resistance and significantly reduce GBM growth and invasion in both established and primary invasive GBM in mice. This study demonstrates the efficacy of SC-based EGFR targeted therapy in GBMs and provides a unique approach with clinical implications.

Structural insights into the role and targeting of EGFRvIII.

The epidermal growth factor receptor (EGFR) is a well-known oncogenic driver in lung and other cancers. In glioblastoma multiforme (GBM), the EGFR deletion variant III (EGFRvIII) is frequently found alongside EGFR amplification. Agents targeting the EGFR axis have shown limited clinical benefits in GBM and the role of EGFRvIII in GBM is poorly understood. To shed light on the role of EGFRvIII and its potential as a therapeutic target, we determined X-ray crystal structures of a monomeric EGFRvIII extracellular region (ECR). The EGFRvIII ECR resembles the unliganded conformation of EGFR, including the orientation of the C-terminal region of domain II. Domain II is mostly disordered, but the ECR structure is compact. We selected a nanobody with preferential binding to EGFRvIII relative to EGFR and structurally defined an epitope on domain IV that is occluded in the unliganded intact EGFR. These findings suggest new avenues for EGFRvIII targeting in GBM.

A bispecific T cell engager recruits both type 1 NKT and Vγ9Vδ2-T cells for the treatment of CD1d-expressing hematological malignancies.

Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory T cells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proinflammatory cytokine production, effector cell expansion, and target cell lysis in vitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.

A Bispecific γδ T-cell Engager Targeting EGFR Activates a Potent Vγ9Vδ2 T cell-Mediated Immune Response against EGFR-Expressing Tumors.

Vγ9Vδ2 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing Vγ9Vδ2 T cells to EGFR-expressing tumors. An EGFR-Vδ2 bispecific T-cell engager (bsTCE) was generated, and its capacity to activate Vγ9Vδ2 T cells and trigger antitumor activity was tested in multiple in vitro, in vivo, and ex vivo models. Studies to explore safety were conducted using cross-reactive surrogate engagers in nonhuman primates (NHP). We found that Vγ9Vδ2 T cells from peripheral blood and tumor specimens of patients with EGFR+ cancers had a distinct immune checkpoint expression profile characterized by low levels of PD-1, LAG-3, and TIM-3. Vγ9Vδ2 T cells could be activated by EGFR-Vδ2 bsTCEs to mediate lysis of various EGFR+ patient-derived tumor samples, and substantial tumor growth inhibition and improved survival were observed in in vivo xenograft mouse models using peripheral blood mononuclear cells (PBMC) as effector cells. EGFR-Vδ2 bsTCEs exerted preferential activity toward EGFR+ tumor cells and induced downstream activation of CD4+ and CD8+ T cells and natural killer (NK) cells without concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs. Administration of fully cross-reactive and half-life extended surrogate engagers to NHPs did not trigger signals in the safety parameters that were assessed. Considering the effector and immune-activating properties of Vγ9Vδ2 T cells, the preclinical efficacy data and acceptable safety profile reported here provide a solid basis for testing EGFR-Vδ2 bsTCEs in patients with EGFR+ malignancies.

Rapid visualization of human tumor xenografts through optical imaging with a near-infrared fluorescent anti-epidermal growth factor receptor nanobody.

Given that overexpression of the epidermal growth factor receptor (EGFR) is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies.

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth.

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.

Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET.

PURPOSE: The ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with (68)Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for (89)Zr immuno-positron emission tomography (PET). METHODS: Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified (68)Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml(-1) gentisic acid, pH 5.5) at 4°C or in human serum at 37°C, a mixture of (67)Ga and (68)Ga was used. Biodistribution and immuno-PET studies of (68)Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using (89)Zr-Df-Bz-NCS-7D12 as the reference conjugate. RESULTS: The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall (68)Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. (68/67)Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (<2% radioactivity loss). In biodistribution studies the (68)Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate (89)Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. CONCLUSION: Via a rapid procedure under mild conditions a (68)Ga-Nanobody was obtained that exhibited high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts. Fast kinetic (68)Ga-Nanobody conjugates can be promising tools for tumour detection and imaging of target expression.

A Bispecific Antibody Antagonizes Prosurvival CD40 Signaling and Promotes Vγ9Vδ2 T cell-Mediated Antitumor Responses in Human B-cell Malignancies.

Novel T cell-based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by Vγ9Vδ2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific Vγ9Vδ2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD40L-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of Vγ9Vδ2 T cells in the presence of CD40+ tumor cells induced potent Vγ9Vδ2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific γδ T-cell engager demonstrated lysis of leukemic cells by autologous Vγ9Vδ2 T cells present in patient-derived samples. Taken together, our CD40 bispecific γδ T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent Vγ9Vδ2 T cell-dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis.

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

The architecture of EGFR's basal complexes reveals autoinhibition mechanisms in dimers and oligomers.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.

Nanobodies in therapeutic applications.

Over the years, many antibodies have been successfully generated to treat patients with life-threatening diseases, most notably cancer. While the first generation of antibodies, originating from mice, caused severe side effects and were relatively inefficient, technological advances have made it possible to obtain fully human antibodies for therapeutic use. 'Heavy-chain only' antibodies have recently been discovered in the blood of camelids. Because of their size, the antigen-binding units of these antibodies comprising only a single Ig fold are called Nanobodies. These antibody fragments have several remarkable features that make them ideal candidates as next-generation cancer therapeutics. Particularly appealing is their ability to simultaneously inhibit various crucial growth factor receptors or their ligands with a single molecule. In addition, they are easy to clone and express on the tip of filamentous phage, which opens the possibility to select for Nanobodies inducing particular biological effects. Nanobodies have potential to become important cancer therapeutics in the near future, displaying unequalled and unprecedented efficacies in treatment.

A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2-T cells.

Though Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in an in vivo mouse xenograft model. Tumor cell lysis was independent of KRAS and BRAF tumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics.

Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform.

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1ß and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.