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Antibody therapy of cancer, AM Scott et al, Nature reviews, 2012
“… What has become evident…(following) the long sought-after cancer-specific antigen group (is that they have) not been found. Rather, antibodies that predominantly bind antigens in cancer cells compared with normal tissues have been found. Despite this lack of absolute specificity for cancer cells, these antibodies with preferential cancer reactivity have among the highest tumor specificity of any targeted therapeutic approach that has yet been defined… Ideally, the target antigen should be abundant and accessible and should be expressed homogeneously, consistently and exclusively on the surface of cancer cells. Antigen secretion should be minimal, as secreted antigens can bind the antibody in the circulation and could prevent sufficient antibody from binding to the tumor. If the desired mechanism of action is ADCC (antibody-dependent cellular cytotoxicity) or CDC (complement-dependent cytotoxicity), then it is desirable that the antigen–mAb complex should not be rapidly internalized so as to maximize the availability of the Fc region to immune effector cells and complement proteins, respectively. By contrast, good internalization is desirable for antibodies or proteins that deliver toxins into the cancer cell and for antibodies the action of which is primarily based on the downregulation of cell surface receptors… Table 1 | Antibody constructs and potential uses in oncology Antibody constructs Examples of targets Potential clinical use scFv CC49, ERBB2 and Ley Imaging and cell targeting Diabody Ley and TAG‑ Imaging and drug delivery Affibody ERBB Imaging and drug delivery Minibody CEA and ERBB Imaging and drug delivery Protein–Fc Angiopoietin 1, angiopoietin 2, VEGFR1 and VEGFR2 Imaging and therapy Intact IgG CD20, CD33, EGFR, ERBB2 and VEGF Imaging therapy and drug delivery IgE and IgM GM Therapy Drug conjugates CD30, CD33 and ERBB2 Therapy Loaded nanoparticles A33, EGFR and transferrin Drug delivery Bispecifics CD19–CD3, EPCAM–CD3 and gp100–CD3 Therapy
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Bi-specific antibodies P. Chames et al 2009, Kontermann et al 2015
The potential of using bispecific antibodies to retarget effector cells toward tumor cells was demonstrated in the 1980s and, several Phase 1 clinical studies were launched in the early nineties. These early bispecific molecules were mainly generated using either of two approaches, chemical cross-linking or hybrid hybridomas or quadromas. Despite some obvious biological effects, none of these approaches led to a significant impact in the clinical course of disease. …More encouraging results were obtained in two clinical trials involving HRS-3/A9, a bispecific F(ab’)2 antibody targeting the CD30 antigen on Hodgkin and Reed-Sternberg cells in patients with Hodgkin disease (HD), and receptor FcγRIII (CD16) expressed by natural killer and macrophages. Two Phase 1 clinical studies performed with this molecule led to one complete remission (CR) and one partial remission (PR) in a group of 15 treated patients…followed by one CR and three PR in a second clinical trial. The construct was the first instance of bsAb treatment leading to a complete remission. However, the low production yield (2.8 g of bsAb obtained from 44 g of IgG) and the high immunogenicity of this bsAb precluded further clinical studies… A different approach, and indeed the most obvious application of bsAb, is T cell retargeting. Cytotoxic T cells are considered the most potent killer cells of the immune system… but do not express Fcγ receptors…Bispecific antibodies directed against a tumor marker and CD3 have the potential to redirect and activate any circulating T cells against tumors. However, T cells have a major drawback. Without the secondary signal given by the interaction between CD28 and one of its ligands (e.g., B7), T cells are not fully activated… bsAbs were thus administered in combination with anti-CD28 antibodies, but the combination yielded mixed results.. Surprisingly, treatment with bsAbs of the most basic format, i.e., chemical cross-linking of full size monoclonal antibodies, yielded some success in clinical studies.. Specifically, the patient’s T cells are purified, expanded and activated in vitro to very large amount (>300 x 109). Next, these cells are incubated with an anti-CD3 x tumor target bispecific antibody, before being administered to the patient..
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Triomabs probably represent one of the most impressive and unexpected success in the field of bispecific antibodies. In 1995, Lindhofer and collaborators published a paper describing a major improvement of the classical quadroma approach to produce bsAbs. By using an original subclass combination (mouse IgG2a and rat IgG2b), they demonstrated a preferential species-restricted heavy/light chain pairing, in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, as well as use of sequential pH elution on protein A to easily separate the desired bsAb from the parental mAb. Surprisingly, the resulting hybrid rat/mouse Fc portion efficiently interacted with activating human Fc receptors (FcγRI and FcγRIII), but not inhibitory ones (FcγRIIB), thereby reaching the goal that other groups had hoped to achieve using human Fc engineering. Catumaxomab, which targets the tumor antigen Epithelial cell adhesion molecule (EpCAM) was the first triomab produced. EpCAM (CD326) is expressed on essentially all human adenocarcinoma, certain squamous cell carcinoma, retinoblastoma and hepatocellular carcinoma. EpCAM is also expressed in normal cells, but is predominantly located in intercellular spaces where epithelial cells form very tight junctions.. By early 2009, the results of a large international Phase 2/3 pivotal study involving 258 patients (ovarian cancer patients with malignant ascites were treated with IP administration of triomab) demonstrated a statistically significant improvement of the primary endpoint, puncture-free survival. Patients receiving catumaxomab had a four-fold increase in puncture-free survival compared to those receiving paracentesis therapy only. TrAb catumaxomab is 1000-fold more potent than HO-3 (monoclonal Ab against EpCAM) in eliciting PBMC-mediated killing of EpCAM-positive carcinoma cells. Comparison of trAb and mAb mediated cytotoxicity against EpCAM-positive tumour cells in vitro. HCT-8 tumour cells and PBMCs were co-cultured at ratios of 10 : 1 in the presence of trAbs catumaxomab (anti-EpCAM × anti-CD3), Bi20 (anti-CD20 × anti-CD3) or mAb HO-3 at the indicated concentrations. Additional approaches with high effector ratios of 50 : 1 were performed for H0-3 and mouse IgG2a isotype control MmT1 (anti-mouse Thy-1.2). After 3 days, tumour cell killing was measured by XTT staining. Alloreactivity of PBMC without antibody was not significant (0–2%). Data points display mean values of four determinations with s.d. Data are representative results from four independent experiments using PBMCs from different donors. The drug is approved for the treatment of malignant ascites in patients with EpCAM-positive cancer if a standard therapy is not available. The usual treatment of malignant ascites is to puncture the peritoneum to let the accumulated fluid drain out. After the puncture, catumaxomab is given as an intraperitoneal infusion. The procedure is repeated four times within about eleven days. It has been shown that puncture free survival can be increased from 11 to 46 days with this treatment
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The New Generation: Recombinant bsAbs
Moderate anti mouse and anti rat responses were seen in the majority of the patients but they do not seem to affect the efficiency of the treatment.26 This is probably due to the extremely small amounts administered to the patients (around 100 μg) compared to 3 g for rituximab (anti-CD20 mAb used in B-cell lymphomas and other B-cell malignancies) i.e. (30,000 fold less), the short duration of the treatment (ten days) and probably to the IP route of administration. However, intravenous (IV) injections will be required for other indications… More of these quadromas are under clinical trials even today! The New Generation: Recombinant bsAbs Tandem scFvs (TaFv) are two scFv fragments linked by an extra peptide linker that is expected to confer good flexibility to each fragment. Numerous studies have demonstrated the potency of these formats in preclinical studies…Surprisingly, no bispecific diabodies have been tested so far in clinical trials. A conventional antibody is depicted in green (light for light chain, dark for heavy chain, blue triangle indicate the glycosylation site) and the derived fragments (shaded areas represent the binding sites). The orange color symbolizes a different specificity. The blue and red shapes represent the DDD2 and AD2 peptides of the dock and lock (DNL) method. All flexible linkers are in grey. bsAb, bispecific antibody; bsFab, bispecific Fab fragment; scFv, single chain Fv fragment; dAb, domain antibody. The Dock and Lock (DNL) method represents a convenient and efficient way to create bispecific antibodies. It relies on the spontaneous association of a dimer of the 45 amino acids peptide DDD2, derived from the regulatory subunit of human cAMP-dependent protein kinase (PKA) with the 21 residues peptide AD2, derived from the anchoring domains (AD) of human A kinase anchor proteins (AKAPs). By reducing the length of the peptide linker between variable domains so that these cannot assemble, it is possible to force the pairing of domains between two different polypeptides, leading to a compact bsAbs called diabody (Db). This format has been improved by adding an extra peptide linker between the two polypeptides in order to further decrease the amount of homodimers, yielding fragments called single chain diabodies (scDb). BiTEs, or bispecific T cell Engagers, are made by fusing an anti-CD3 scFv to an anti-TAA (tumor associated antigen) scFv via a short five residue peptide linker (GGGGS).
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We propose here a solution for the correct assembly of four different chains leading to a bispecific IgG-like antibody. Light-chain mispairing occurs because the molecular architectures of the heterodimerization interfaces between the variable heavy (VH) and the variable light (VL) and between the constant heavy 1 (CH1) and the constant light (CL) domains in both arms of an antibody are identical. For the bispecific antibody, we have rearranged heavy- and light-chain domains in one arm of the antibody to make these interfaces different. Starting from the two arms of the theoretically desired bispecific antibody (Fig. 1A), we design four chains as follows (Fig. 1B). On one side, we leave heavy chain 1 and light chain 1 unmodified. On the opposite side, we design a new “heavy” chain 2 consisting of an Fc part and the Fab of the original light chain. As the new “light” chain 2 we use the heavy chain domains VH and CH1. Because the sequence of the modified heavy chain now is crossing over between light- and heavy-chain domains, we use the term “crossover” for this kind of domain interchange and the term “CrossMab” for antibodies based on this technology. Heterodimerization of the two heavy chains is achieved by using the KiH method. As a consequence of this domain rearrangement, associations between unrelated partners can no longer occur. The new “light” chain 2 on the crossover side consists of heavy-chain domains only; thus it cannot assemble with the remaining original heavy chain 1. On the other hand, the original light chain 1 on the unmodified side cannot interact with the new “heavy” chain 2 on the crossover side, because both partners contain the same light-chain heterodimerization interfaces, which do not interact. We thus obtain a bispecific antibody (Fig. 1C) with correct light-/heavy-chain pairing in both Fabs and almost no deviation from the original IgG. In this “CrossMabFab” the chain crossover has been applied on a part of the antibody that contains two heterodimerization interfaces (VH-VL and CH1-CL). However, a differentiation of the light-chain/heavy-chain associations in both arms also can be achieved by exchanging only one of these heterodimerization pairs. Replacement of VH by VL and vice versa leads to a “CrossMabVH-VL” (Fig. 1D); interchange of CH1 and CL results in a “CrossMabCH1-CL” (Fig. 1E). In total, we obtain three different CrossMab formats. This approach is generic, because it can be applied to any two existing antibodies without further optimization of the individual protein sequences. Schematic diagram of the Fab domain exchange resulting in the generation of a bispecific antibody when combined with the KiH technology. Schematic diagram of the Fab domain exchange resulting in the generation of a bispecific antibody when combined with the KiH technology. Dark colors indicate heavy-chain domains. Light colors indicate light-chain domains. (A) Both arms of the intended bispecific antibody. (B) Design of the four chains of the bispecific antibody. Heavy-chain heterodimerization is achieved by use of the KiH technology. (C) Crossover of the complete VH-CH1 and VL-CL domains. (D and E) Crossover of only the VH and VL domains (D) or the CH1 and CL domains (E) within the Fab region of one half of the bispecific antibody. Wolfgang Schaefer et al. PNAS 2011;108: ©2011 by National Academy of Sciences
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Bispecific Ang-2–VEGF CrossMabCH1-CL inhibited tumor growth of Colo205 tumors and VEGF-induced corneal angiogenesis more potently compared to the monotherapies of bevacizumab (Avastin) and LC06 or their combination. Bispecific Ang-2–VEGF CrossMabCH1-CL inhibited tumor growth of Colo205 tumors and VEGF-induced corneal angiogenesis more potently compared to the monotherapies of bevacizumab and LC06 or their combination. (A) Treatment with Ang-2–VEGF CrossMabCH1-CL showed significant inhibition of s.c. Colo205 tumor growth over time (*P < 0.05 compared with control group, by student's t test). Ang-2–VEGF CrossMabCH1-CL, bevacizumab, LC06, and control IgG (human IgG) were administered i.p. (n = 10 mice per group). Treatment started at day of randomization (study day 12) with 10 mg/kg once weekly (indicated by arrows). (B) Analysis of neovascularization in the mouse corneal angiogenesis model. Systemic treatment with Ang-2–VEGF CrossMabCH1-CL (10 mg/kg) resulted in an inhibition of corneal angiogenesis to background levels (*P < 0.05 compared with control group, by student's t test). Wolfgang Schaefer et al. PNAS 2011;108: ©2011 by National Academy of Sciences
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Christian Klein, head of oncology programs at Roche's Glycart AG subsidiary, told SciBX that the first piece of the puzzle was solved by Genentech Inc. almost 15 years ago. “A major breakthrough came in 1997 when Genentech developed the Knob-into-Hole [KiH] technology, which allows the heterodimerization of two different heavy chains. However, the problem remained that you would still get the wrong light chain associations,” he said. Genentech is now a unit of Roche. KiH technology works by replacing specific amino acids at the heavy chain dimerization interface so that two distinct heavy chain fragments heterodimerize with each other instead of homodimerizing with themselves. However, the two light chain fragments would still be capable of pairing indiscriminately with either heavy chain, leading to unwanted antibody products. Roche has now solved that problem with its CrossMab technology, and the company provided proof of concept by joining its anti-VEGF antibody Avastin bevacizumab with LC06, an anti–angiopoietin 2 (ANG2; ANGPT2) antibody. ANG2 is an angiogenesis-promoting growth factor that is upregulated in some tumors.
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