Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody

The pancaner molecule CD276 (B7-H3) is an attractive target for antibody based therapy. We identified from a large (10¹¹) phage-displayed single-chain variable fragment (scFv) library, a fully human antibody, B11, which bound with high avidity (K_D=0.4 nM) to CD276. B11 specifically bound to the V1/V2 domain of CD276 and competed with the antibody 8H9 (Omburtamab). It was used to design an IgG-format bispecific T cell engager B11-BiTE, which was more effective than 8H9-BiTE in 14 different cancer cell lines. B11-BiTE also exhibited strong ADCC/ADCP. Therefore, the fully human B11-BiTE is a promising candidate for treatment of tumors expressing CD276.     

Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody

The pancaner molecule CD276 (B7-H3) is an attractive target for antibody based therapy. We identified from a large (10¹¹) phage-displayed single-chain variable fragment (scFv) library, a fully human antibody, B11, which bound with high avidity (K_D=0.4 nM) to CD276. B11 specifically bound to the V1/V2 domain of CD276 and competed with the antibody 8H9 (Omburtamab). It was used to design an IgG-format bispecific T cell engager B11-BiTE, which was more effective than 8H9-BiTE in 14 different cancer cell lines. B11-BiTE also exhibited strong ADCC/ADCP. Therefore, the fully human B11-BiTE is a promising candidate for treatment of tumors expressing CD276.     

Non-T cell killing of mammary tumor cells by spleen cells: secretion of antibody and recruitment of cells.

Both T cell-mediated killing and non-T cell-mediated killing of target MTV-induced mammary tumor cells can be detected in microcytotoxicity assay tests of spleen cells from mice immunologically responsive to either the histocompatibility antigens or the virus-associated antigens of the target cells. The non-T cell-mediated cytotoxicity is antibody-dependent; otherwise inactive cells (null cells) can be recruited to activity by target cell-specific factors obtained from the supernatant of short-term cultures of sensitized B cells or provided by the introduction of a small number of sensitized B cells to the wells of the assay plate.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.     

A new class of bispecific antibodies to redirect T cells for cancer immunotherapy

Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK^TM (DNL^TM) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls.     

A new class of bispecific antibodies to redirect T cells for cancer immunotherapy

Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK^TM (DNL^TM) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls.     

Antibody-mediated targeting of human single-chain class I MHC with covalently linked peptides induces efficient killing of tumor cells by tumor or viral-specific cytotoxic T lymphocytes

Soluble forms of human MHC class I HLA-A2 were produced in which the peptide binding groove was uniformly occupied by a single tumor or viral-derived peptides attached via a covalent flexible peptide linker to the N terminus of a single-chain -2-microglobulin-HLA-A2 heavy chain fusion protein. A tetravalent version of this molecule with various peptides was found to be functional. It could stimulate T cells specifically as well as bind them with high avidity. The covalently linked single chain peptide-HLA-A2 construct was next fused at its C-terminal end to a scFv antibody fragment derived from the variable domains of an anti-IL-2R subunit-specific humanized antibody, anti-Tac. The scFv–MHC fusion was thus encoded by a single gene and produced in E. coli as a single polypeptide chain. Binding studies revealed its ability to decorate Ag-positive human tumor cells with covalent peptide single-chain HLA-A2 (scHLA-A2) molecules in a manner that was entirely dependent upon the specificity of the targeting Antibody fragment. Most importantly, the covalent scHLA-A2 molecule, when bound to the target tumor cells, could induce efficient and specific HLA-A2-restricted, peptide-specific CTL-mediated lysis. These results demonstrate the ability to generate soluble, stable, and functional single-chain HLA-A2 molecules with covalently linked peptides, which when fused to targeting antibodies, potentiate CTL killing. This new approach may open the way for the development of new immunotherapeutic strategies based on antibody targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.Kfir Oved and Avital Lev contributed equally to this work     

Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells

We have used two monoclonal antibodies (Mab) to the L3T4 antigen to reexplore the role of this molecule in the process of T cell activation. Both Mab (Gk1.5 and 2B6) were capable of inhibiting Con A-induced IL 2 production by a number of antigen-specific T cell hybridomas in an assay system that was free of major histocompatibility complex (MHC) class II antigen-bearing cells. The inhibition produced by the anti-L3T4 Mab was specific, because other Mab to cell surface antigens expressed on the hybridomas were without inhibitory effects. These studies rule out the possibility that the mechanism of inhibition by anti-L3T4 in this model is mediated by blocking interaction of L3T4 with MHC class II products. Taken together, these results and those of other groups of investigators, are most compatible with a dual function for L3T4 in T cell activation. L3T4 might first interact with MHC class II molecules or other molecules on target or accessory cells; L3T4 would subsequently transmit a signal that would regulate the activation process. Mab to L3T4 might exert inhibitory effects at one or both of these steps.     

Effect of concanavalin A on the killing of tumor cells by antibody and complement.

Concanavalin A (Con A) was found to inhibit the killing of antibody-sensitized line-1 tumor cells (TA) by guinea pig complement (GPC) but not by human complement (HuC). Other plant lectins (wheat germ, leucoagglutinin, and pokeweed mitogen) were also tested but Con A was the only lectin found to inhibit antibody-GPC-mediated killing. The inhibitory effect of Con A was observed when the GPC was mixed with Con A or when the antibody-sensitized cells were pretreated with Con A (TA-Con A) before the addition of GPC. The effect could be reversed by treatment of such cells with alpha-D-methylglucopyranoside or by incubation at 37 degrees C for approximately 2 hr. Con A appeared to act by preventing the binding of the first component of GPC (GPC1) to antibody-sensitized tumor cells. Differences in the binding of the first component of HuC (HuC1) and GPC1 to TA-Con A suggested that a difference in the binding site for HuC1 and GPC1 might exist. There was no difference in the number of GPC1 molecules fixed to antibody-sensitized sheep erythrocytes (EA) or EA treated with Con A in experiments using the same antibody as used with the tumor cells and the same Con A preparation. It would consequently appear that the inhibitory effect of Con A on the binding of GPC1 to TA is not due solely to an interaction of Con A with the antibody.     

Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody,inhibits tumor progression and angiogenesis

Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been approved for cancer treatment. However, VEGF inhibitors alone were shown to promote tumor invasion and metastasis by increasing intratumoral hypoxia in some preclinical and clinical studies. Emerging reports suggest that Delta-like ligand 4 (Dll4) is a promising target of angiogenesis inhibition to augment the effects of VEGF inhibitors. To evaluate the effects of simultaneous blockade against VEGF and Dll4, we developed a bispecific antibody, HD105, targeting VEGF and Dll4. The HD105 bispecific antibody, which is composed of an anti-VEGF antibody (bevacizumab-similar) backbone C-terminally linked with a Dll4-targeting single-chain variable fragment, showed potent binding affinities against VEGF (K_D: 1.3 nM) and Dll4 (K_D: 30 nM). In addition, the HD105 bispecific antibody competitively inhibited the binding of ligands to their receptors, i.e., VEGF to VEGFR2 (EC₅₀: 2.84 ± 0.41 nM) and Dll4 to Notch1 (EC₅₀: 1.14 ± 0.06 nM). Using in vitro cell-based assays, we found that HD105 effectively blocked both the VEGF/VEGFR2 and Dll4/Notch1 signaling pathways in endothelial cells, resulting in a conspicuous inhibition of endothelial cell proliferation and sprouting. HD105 also suppressed Dll4-induced Notch1-dependent activation of the luciferase gene. In vivo xenograft studies demonstrated that HD105 more efficiently inhibited the tumor progression of human A549 lung and SCH gastric cancers than an anti-VEGF antibody or anti-Dll4 antibody alone. In conclusion, HD105 may be a novel therapeutic bispecific antibody for cancer treatment.     

Mechanism of cytolytic T lymphocyte killing of a low class I-expressing tumor

Many tumors have been shown to express minimal levels of class I MHC Ag, which makes them more resistant to recognition and lysis by cytolytic T lymphocytes. Line 1, a BALB/c spontaneous lung carcinoma, normally expresses very low levels of class I Ag, but expression can be increased 50-fold by treatment with agents such as DMSO or IFN-gamma. Because class I Ag serve as restricting elements for cytolytic T cell recognition of tumor Ag, we wished to determine if cytotoxic T lymphocytes could play a role in the immune response to this type of class I low, but inducible, tumor. After immunization in vivo and restimulation of splenic cells in vitro we were able to generate T cell clones that lysed line 1 cells induced to express high levels of class I, but did not lyse uninduced, low class I expressing line 1 cells in short term (6-h) 51Cr release assays. Paradoxically, incubation of the T cells with uninduced class I low line 1 cells for a few days resulted in complete destruction of the tumor cells. We demonstrate that the T cells, stimulated by the tumor cells, produce IFN-gamma, which in turn induces class I expression on the line 1 cells making them susceptible to lysis by the T cell clone. This suggests that a positive feedback reaction can occur in generating a response to this and perhaps other inducible tumor cell lines.     

Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.     

T cell activation and retargeting using staphylococcal enterotoxin B and bispecific antibody: An effective in vivo antitumor strategy

 The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with staphylococcal enterotoxin B (SEB) and retargeting with antitumor×anti-CD3 F(ab′)₂ bispecific antibodies (bsAb). All studies were performed in C3H/HeN mice using syngeneic tumor cell lines. For survival studies, mice were injected intravenously on day 0 with CL62 (a p97-transfected clone of the K1735 murine melanoma tumor). Day-3 treatments included saline (control), SEB (50 γg intraperitoneal) with or without bsAb (5 μg i.v.). Cured mice, surviving beyond 60 days, were rechallenged with subcutaneous CL62, K1735, or a nonmelanoma control, AG104. SEB activation studies were performed with pulmonary tumor-infiltrating lymphocytes isolated from 10-day established CL62 tumors. Maximal tumor-infiltrating lymphocyte cytotoxicity was demonstrated 24 h following SEB injection, therefore bsAb treatments were administered 24 h after SEB. When survival was examined at 60 days, there were significantly more survivors in the group receiving SEB plus bsAb (70%) compared to the group receiving SEB alone (30%), and the controls (0%) (P=0.02 and P<0.01, respectively). Mice cured of CL62 using SEB alone or with bsAb demonstrated equal immunity to CL62, however, mice treated with SEB plus bsAb were more often immune to the p97^– parental cell line, K1735(P=0.001). Ag104 consistently grew in all mice. Results of these studies demonstrate that SEB plus bsAb can be effective, not only in curing tumors but also in providing protective immunity against targeted and nontargeted tumor antigens. Accepted: 14 October 1997     

T cell activation. II. Activation of human T lymphoma cells by cross-linking of their MHC class I antigens

The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cai2+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Cai2+ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when Cai2+ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CD3-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules.     

Requirements for suppressor cell activation. Role of accessory cells

In the 4-hydroxy-3-nitrophenyl acetyl (NP) suppressor system, third order suppressor cells (Ts3) subset of suppressor cells is generated after Ag priming, but, in order to express suppressor activity, these cells need to be further activated or triggered with a specific second order suppressor factor. By in vitro activation of Ts3-containing lymph node cells or a pTs3 hybridoma we now show that macrophages are also required for Ts3 activation. In addition, we demonstrate that IJ genetic restrictions control this activation process. Furthermore, we directly demonstrate Ts3 activation using cloned macrophage hybridoma cells. To further investigate the interactions between Ts3 cells and the accessory cells involved in their activation, we attempted to block the second order suppressor factor mediated activation of Ts3 cells with antibodies. The activation of Ts3 cells can be blocked by the addition of anti-IJ, anti-IJ idiotype or anti-NPb idiotype antibodies, but not by anti-CD8, anti-IA, or anti-IE antibodies. Anti-IJ mAb blocked Ts3 activation at the lymphocyte level whereas anti-IJ idiotype blocked activation at the accessory cell level. Finally we tested, whether these antibodies can also directly activate primed Ts3 cells. We demonstrate that cross-linked anti-IJ, anti-NPb and anti-CD3 antibodies can activate Ts3 cells. The results are discussed in terms of receptor-ligand structures on Ts and accessory cells which are required for the activation of Ts3 cells.     

Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein.

Bispecific antibodies directed against tumour associated antigens and the T cell receptor component CD3 for recruitment and tumour targeted activation of T cells represent a novel class of highly specific immunotherapeutics for cancer. We here describe the construction, eukaryotic expression and in vitro functional activity of a new T cell activating bispecific reagent, termed TTS for T cell targeting to the tumour stroma, comprised of a CD3 specific single chain antibody derivative (scFv) fused C-terminally to a 'fibroblast activation protein' (FAP) specific scFv that targets cytotoxic effector cells to FAP. FAP is highly expressed in the vascularised tumoural stroma of most lung, breast and colon carcinomas. It thus represents a selectively tumour associated, yet common marker of many solid tumours and is a potentially ideal candidate marker for efficient targeting of immune effector cells.     

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

T-cell redirecting bispecific antibodies (bsAbs) or antibody-derived agents that combine tumor antigen recognition with CD3-mediated T cell recruitment are highly potent tumor-killing molecules. Despite the tremendous progress achieved in the last decade, development of such bsAbs still faces many challenges. This work aimed to develop a mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) modeling framework that can be used to assist the development of T-cell redirecting bsAbs. A Target cell-Biologics-Effector cell (TBE) complex-based cell killing model was developed using in vitro and in vivo data, which incorporates information on binding affinities of bsAbs to CD3 and target receptors, expression levels of CD3 and target receptors, concentrations of effector and target cells, as well as respective physiological parameters. This TBE model can simultaneously evaluate the effect of multiple system-specific and drug-specific factors on the T-cell redirecting bsAb exposure–response relationship on a physiological basis; it reasonably captured multiple reported in vitro cytotoxicity data, and successfully predicted the effect of some key factors on in vitro cytotoxicity assays and the efficacious dose of blinatumomab in humans. The mechanistic nature of this model uniquely positions it as a knowledge-based platform that can be readily expanded to guide target selection, drug design, candidate selection and clinical dosing regimen projection, and thus support the overall discovery and development of T-cell redirecting bsAbs.