A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma.

T-cell-mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein-coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).

Selection and maturation of antibodies by phage display through fusion to pIX.

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM.

Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage.

Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins.

De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins.

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V(L) and three V(H) domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V(H) by keeping V(L) in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V(H) diversity was sufficient for isolating Fabs to most antigens, whereas variability in V(L) was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V(L) diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.

Generation, characterization and biological activity of CCL2 (MCP-1/JE) and CCL12 (MCP-5) specific antibodies.

The human CCL2 chemokine is implicated in many chronic inflammatory conditions. In the mouse, there are two CCL2 homologues, CCL2 (MCP-1/JE) and CCL12 (MCP-5). Both are potent monocyte chemoattractants and bind to and activate the same receptor, CCR2. The overlapping activities of these chemokines complicate the design of mouse model studies that are intended to mimic human disease. To study the roles of CCL2 and CCL12, we generated neutralizing antibodies specific to each chemokine. Consistent with binding and affinity analyses, the antibodies specifically inhibited CCL2- or CCL12- mediated Ca(2+) mobilization in THP-1 cells. When tested in nude mice bearing human PANC-1 pancreatic tumor cells in Matrigel plugs, CCL2 and CCL12 antibodies potently inhibited tumor angiogenesis, indicating that both CCL2 and CCL12 may contribute to tumor angiogenesis.

Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies.

Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity. A phage display Fab library was selected on recombinant F antigen in the presence of three representative antibodies recovered in the unblocked and subsequent single-blocked panning procedures. Restriction endonuclease fingerprinting of 13 F+ clones revealed seven unique Fabs. DNA sequence analysis of five of these clones revealed five new light chains in combination with different heavy chains, three of which were very similar or identical to Fabs previously isolated from this library. The blocking antibodies did not compete with the new Fabs, demonstrating effective masking of their binding sites in the panning procedure. Conversely, these Fabs did show variable inhibition of two of the blocking antibodies suggesting a close proximity or interdependence of their epitopes. One of the antibodies did inhibit virus infection, albeit with modest potency. These results demonstrate that epitope-blocked panning is a self-progressing approach to retrieving diverse antibodies from phage libraries.