An FDA oncology analysis of CD3 bispecific constructs and first-in-human .. The following information was collected for each IND from FDA/. Introduction. Blinatumomab (Blincyto) is a bispecific T-cell engager antibody construct that binds to 4 Are all required (*) and requested IND. The company just announced that the FDA has cleared the IND application for a humanized bispecific GD2 antibody. According to the release, it is anticipated.
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Indian Association for the Cultivation of Science. Chemically programmed bispecific antibodies biAbs endow target cell-binding small molecules bispecicic the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells.
Two different antibody technologies were merged together to make a novel chemically programmed biAb.
Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy. Chemically programmed bispecific antibodies biAbs 6 that exert cytotoxicity by binding to tumor cells with one arm and by simultaneously recruiting and activating tumor cell-lysing T cells with the other arm are an emerging category of next-generation antibody drugs for cancer therapy 1 — 4.
Merging this promising platform with the concept of chemically programmed antibodies 5we and others recently developed chemically programmed biAbs that recognize tumor cells with a variable small molecule component and that recruit and activate T cells with a generic antibody component 6 — 8.
Chemically programmed biAbs are more versatile than conventional biAbs as they only require the cloning, expression, and purification of a single protein Fig. Further, to target a variety of different tumor cell surface antigens, chemically programmed biAbs can make use of a wealth of small molecules derived from chemical libraries or from structure-based design campaigns, linking advances in both immunology and chemistry for the benefit of cancer patients. As depicted in Fig. However, our current report is the first to utilize h38C2 as an antibody component in chemically programmed biAbs designed to recruit and activate endogenous T cells in bispedific patients.
For combination of h38C2 bipsecific v9, a number of biAb formats exist 2. Among these, the BiTE for Bi specific T -cell E ngager format, which combines two single chain Idn scFv modules linked by a polypeptide linker, is of particular interest.
These favorable features of the BiTE format are attributed to: The success of the BiTE format triggered the search for intellectual property space among biAb formats of similar size and valence For example, a potentially competing format coined DART for D ual- A ffinity R e- T argeting is based on the so called diabody format that separates cognate variable domains of heavy and light chains of the two antigen or hapten binding specificities on two separate polypeptide chains Whereas the two polypeptide chains associate non-covalently in the diabody format, the DART format provides additional stabilization via a C-terminal disulfide bridge Figs.
DARTs can be produced in high quantity and quality and have exceptional stability in both formulation buffer and human serum The more rigid configuration of the DART format, where there is limited flexibility between the two antigen or hapten binding specificities, likely accounts for these improved features 23 Folate was used as a representative tumor-cell targeting small molecule as FOLR1 is a clinically investigated target for both mAbs and small molecules in cancer therapy in general and in the treatment of ovarian cancer in particular 27 — Our findings support the notion of broad therapeutic utility of chemically programmed biAbs at the interface of immunology and chemistry.
BiAbs in DART format are comprised of two polypeptides that are linked at their C termini via a disulfide bridge, where each polypeptide contains one of two cognate variable light and heavy chain domains that form the antigen or hapten binding inc 25 Fig.
A His tag H 6 id included on one of the polypeptides to facilitate purification and detection. The amino acid sequences of hv-L and hv-H are given in the supplemental information with the reactive lysine residue of h38C2 highlighted. These two different configurations were initially pursued in parallel as they may have different antigen or hapten binding properties depending on the involvement of bislecific N termini.
The four polypeptide encoding sequences were generated by custom synthesis and cloned into mammalian expression vector pCEP4 under control of a CMV promoter. AbiAbs in DART format are comprised of two polypeptides that are linked at their C termini via a disulfide bridge, where each polypeptide contains one of two cognate variable light white and heavy chain gray domains that form the antigen or hapten binding site.
This reversible covalent conjugation top is innd by imine-enamine tautomerism. The variable domains on each polypeptide are fused with a short polypeptide G 3 SG 4 that favors diabody over scFv formation. The two polypeptide expression cassettes were cloned under the control of a CMV promoter into mammalian expression vector pCEP4 for transient co-transfection into HEK cells. The reactive Lys residue of h38C2 is indicated in red.
Note that the hv-L configuration has free light chain N termini, whereas configuration hv-H displays free heavy chain N termini. SPsignal peptide. All amino acid sequences are given in the supplemental information. The amino acid sequences of fv-L and fv-H are given in the supplemental information. Note that farletuzumab recognizes a FOLR1 epitope that does not overlap with the folate binding site In addition, the DARTs showed similar binding to CD3-expressing human T-cell line Jurkat by flow cytometry, confirming the integrity of their v9 antigen binding site Fig.
Correct assembly of the chemically programmable DART was further corroborated by detecting catalytic activity of the chemically programmable DARTs but not the conventional DARTs, indicating the functional formation of the h38C2 hapten binding site which involves both variable domains Fig.
A protein marker in kDa was run in the center lane. Ccatalytic activity of purified DARTs hv-L top ; open red squares and hv-H bottom ; open red squares measured with the fluorogenic retro-aldol substrate methodol. Chemical programming with compound 1 eradicated the catalytic activity of both DARTs solid red squares. The background signal of the secondary reagents alone is shown in pale blue. The background signal of the secondary reagents alone is shown in gray.
The results are representative of three experiments. Conjugation of 1 to hv-L and hv-H completely eradicated their catalytic activity Fig. As shown in Fig. The formation of red- and blue-stained cell aggregates was quantified by flow cytometry.
Bresults from all three experiments were plotted as percentage of double positive events among all events following subtraction of the vda observed with unprogrammed DART hv-L. Next, we compared the chemically programmed and conventional DARTs with respect to mediating cytotoxicity in the presence of ex vivo expanded primary human T cells. Unlike their equivalent potency toward OVCAR3 cells and consistent with the noted differences in cell binding and crosslinking capability, we detected significantly lower in vitro cytotoxicity of the chemically programmed compared with the conventional DART toward IGROV1 cells.
T ratio of Luminescence measured after incubation of effector and target cells in the absence of DARTs was subtracted.
IGROV1 xenografts are established models of ovarian cancer with peritoneal carcinomatosis and ascites mimicking the human disease 33 To allow in vivo bioluminescence imaging, we first stably transduced IGROV1 cells with a lentiviral vector encoding firefly luciferase ffluc. Prior to treatment, the cda tumor xenograft was allowed to establish and grow for 6 days.
This is in contrast to a previous study with a FOLR1-targeting chemically programmed biAb, where tumor dfa and T cells at 1: The fourth cohort received PBS alone. The cohorts were treated with a total of 10 daily i. The three control groups, i. No weight loss or other obvious signs of toxicity were bispwcific during the treatment with the DARTs Fig. innd
An FDA oncology analysis of CD3 bispecific constructs and first-in-human dose selection.
Shown are bioluminescence images from day 23 for all 30 animals. A total of 30 NSG mice were i. The animals received a total of ibd daily day 6 to 15 i.
Astarting on day 2, the animals were imaged every 3—4 days as indicated. Treatment time points are indicated by black arrows. Bstarting on day 0, the animals were weighted on the indicated days.
BiAbs are an emerging class of antibody drugs for cancer therapy. Of particular interest are biAbs that kill tumor cells by recruiting and activating endogenous T cells of the cancer patient. As such, these biAbs are comprised of a variable antibody module with specificity for a tumor cell surface antigen and a generic antibody module that has specificity for an activating T-cell receptor, typically CD3.
Its favorable preclinical data along with its successful clinical translation, prompted us to explore the DART format for the generation of chemically programmed biAbs. For this concept, we paired two generic antibody modules, one with a covalent hapten binding site and the other with specificity for CD3. The antibody module with the covalent hapten binding site, h38C2, replaces the variable antibody module targeting tumor cell surface antigens.
Through covalent conjugation of hapten derivatized small molecules that target tumor cell surface antigens to a unique reactive lysine residue in the h38C2 antibody module, our DART can be chemically programmed to acquire virtually any specificity mediated by a small molecule. In fact, h38C2 IgG1 has been previously utilized for chemical programming 912 and translated as so called CovX-Bodies 39 to phase I and II clinical trials enrolling hundreds of cancer and type II diabetes mellitus patients 5.
It functions as a high affinity cellular entry receptor for folate a. Tumor cells, activated macrophages, and proximal tubule epithelial cells of the kidney express FOLR1 to direct effective uptake of folate, whereas most other cell types obtain folate via the low affinity reduced folate carrier SLC19A1 or the proton-coupled folate transporter SLC46A1. On the apical surface of the proximal tubule of the kidney FOLR1 serves as a salvage receptor for folate transport from the nascent urine back to the blood.
Nonetheless, despite rigorous evaluation, kidney toxicity was not observed in phase I clinical trials with FOLR1-targeting small molecules 42 and mAbs 43likely due to rapid transcytosis and inaccessibility, respectively.
The two most advanced FOLR1-targeting drugs are the small molecule vintafolide, which is a conjugate of folate and vinblastine, and the humanized mouse anti-human FOLR1 mAb farletuzumab. After proving generally safe and well tolerated in phase I and II clinical trials, vintafolide and farletuzumab entered phase III clinical trials for the therapy of ovarian cancer. However, both failed to statistically improve progression free survival. This disappointing outcome provides a strong incentive for the development of more potent FOLR1-targeting therapeutics.
In fact, several previous studies have explored strategies for initially random 45 — 47 and eventually site-specific 68 conjugation of folate derivatives to CD3- or TCR-targeting mAbs.
Our in vitro and in vivo data show that both potently kill ovarian cancer cells through recruitment and activation of human T cells. This higher activity appeared to be directly related to the stronger binding and cross-linking ability of the conventional DART and can be attributed to their distinct affinities, epitopes, and internalizations. Importantly, it is conceivable that modifications to the small molecule component of our chemically programmed DART will further improve its performance.
For example, synthetic folate derivatives with two folate moieties spaced by a flexible linker would afford an avidity gain through bivalent binding of FOLR1.
Indeed, we previously showed that a chemically programmed biAb with a bivalent small molecule component was outperforming its counterpart with a monovalent small molecule component 6. Notably, our generic design enables rapid lead optimization as a series of synthetic folate derivatives can be utilized to chemically program the same protein without any need for further cloning, expression, and purification. Although it is highly desirable to reduce treatment to weekly or biweekly i.
A key advantage of chemically programmed antibodies and biAbs is their generic design that not only enables to confine lead optimization to the small molecule component but also permits targeting a virtually unlimited number and variety of antigens with a single protein 5.
Several of these have already been investigated in phase I and II clinical trials, including a bispecific peptide Collectively, our chemically programmable DARTs afford a versatile plug-and-play platform with broad utility in cancer immunotherapy.
Y-mAbs Therapeutics, Inc(NASDAQ:YMAB) Gets the Bispecific IND in Play
Methodol was synthesized as described Sequences of the constructs were based on published or patented amino acid sequences. Quantitation of the chemical programming efficacy, i. Catalytic activity was analyzed using methodol 56 as described previously Cytotoxicity was measured using CytoTox-Glo Promega following the manufacturer’s protocol with minor modifications.