Laurence J.N. Cooper

Variable domain-identical antibodies exhibit IgG subclass-related differences in affinity and kinetic constants as determined by surface plasmon resonance

We have analysed the binding of variable domain-identical mouse monoclonal antibodies (mAb) of the IgG3, IgG1 and IgG2b subclasses, as well as F(ab)2 fragments derived from the IgG3 and IgG1 mAb, to a multivalent glycoprotein target. Using a biosensor device (BIAcore, Pharmacia Biosensor) that measures the mass of the antibody (or other receptor molecule) deposited on a sensor chip displaying the relevant epitopes, we found that the IgG3 mAb binds more effectively than the other antibody species at a high but not a low epitope density. The greater functional affinity associated with the IgG3 mAb, at high epitope density, was correlated with both slower dissociation rate constants and faster association rate constants in comparison with the IgG1 and IgG2b mAb and the F(ab)2 fragments derived from the IgG3 and IgG1 mAb. Evidence for slower dissociation kinetics for the IgG3 mAb versus the IgG1 and IgG2b mAb was also obtained by ELISA and flow cytometry. These results demonstrate that: (1) differences in heavy chain constant (CH) domains can significantly influence apparent functional affinity for multivalent antigen, as determined without the use of covalently modified primary or secondary antibodies; (2) differences in CH domains can alter both association and dissociation rate constants for interactions between IgG antibodies and multivalent antigen; and (3) these effects of CH domains depend on epitope density. The effect of constant region differences on the apparent association rate constants suggests new approaches for achieving better binding or functional effectiveness through antibody engineering.

Intermolecular cooperativity: a clue to why mice have IgG3?

Mouse IgG3 subclass antibodies predominate in humoral responses to bacterial polysaccharide antigens. The reasons for this isotype restriction are not fully understood. Here, Neil Greenspan and Laurence Cooper propose that intermolecular cooperativity, a novel mechanism of antibody binding, may help to explain the preferential expression of IgG3 antibodies in these responses.

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Clinical application of Sleeping Beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Cooperative binding of two antibodies to independent antigens by an Fc-dependent mechanism.

Binding of a murine N‐acetylglucosamine (GlcNAc)‐specific IgG3 monoclonal antibody to a solid phase expressing GlcNAc determinants and phosphocholine (PC) determinants is enhanced by IgG3, but not IgG2b, PC‐specific monoclonal antibody. The cooperative binding requires an intact Fc region on the GlcNAc‐specific monoclonal antibody and is hypothesized to result from Fc‐Fc association. Although the in vivo relevance of this phenomenon requires further study, intermolecular cooperativity in binding of antibody to multivalent antigens, such as bacterial cell wall antigens, could represent an adaptive mechanism for antibodies expressing low intrinsic affinities for highly repeated epitopes. Furthermore, the ability of antibodies of distinct specificity to participate in cooperative binding offers, at least in principle, new approaches to optimizing antibody targeting.—Greenspan, N. S.; Dacek, D. A.; Cooper, L. J. N. Cooperative binding of two antibodies to independent antigens by an Fc‐dependent mechanism. FASEB J. 3: 2203‐2207; 1989.

Cooperative binding by mouse IgG3 antibodies: implications for functional affinity, effector function, and isotype restriction.

ConclusionsThe studies we have reviewed on cooperative antibody binding by mouse IgG3 antibodies have expanded our conception of the functional consequences of structural diversity in immunoglobulin heavy chain C domains. We now know that, in the context of interactions between antibodies and multivalent antigens, CH domain differences can influence both the functional affinities characterizing those interactions and the abilities of the antibodies to discriminate among targets of varying epitope density [7, 8]. This relationship between CH domains and antibody binding illustrates the more general point, discussed in detail elsewhere [16, 18], that antibody-antigen interactions cannot be adequately characterized by focusing solely on the intermolecular (epitope-paratope) interface. Furthermore, CH domain differences can indirectly influence effector functions, such as the activation of the classical complement pathway, through effects on noncovalent Fc-Fc interactions. Given all that is currently known about the biology of IgG3 antibody responses, the occurrence of cooperative binding by IgG3 antibodies also supports the view that some of the differences among IgG subclasses reflect adaptations to particular types of immunological challenges. Future studies on cooperative antibody binding will be directed at: (1) elucidating the molecular basis of the cooperative binding of mouse IgG3 antibodies, and possibly related antibodies from other species, (2) exploring the physiological and pathophysiological consequences of the phenomenon, and (3) exploiting the diagnostic and therapeutic potential of mAb, or mAb-derived Fab or Fv fragments, endowed with the ability to self-associate through protein engineering [14, 17, 22, 30, 41, 44, 55].

Complementarity, specificity and the nature of epitopes and paratopes in multivalent intereactions

Structural elements of an antibody (Ab) or antigen (Ag) distant from the actual sites mediating contact between Ab and Ag can exert substantial influence on binding to, and discrimination among, multivalent targets. Consequently, multivalent molecules that express the same number of identical binding sites, but that differ in other structural features, can exhibit differences in their ability to discriminate between multivalent ligands. Here, Neil Greenspan and Laurence Cooper review evidence for these effects, and explore implications of the conclusion that effective specificity in multivalent interactions is not completely determined by the degree of complementarity between epitopes and paratopes.

Plasma circulating-microRNA profiles are useful for assessing prognosis in patients with cytogenetically normal myelodysplastic syndromes.

Myelodysplastic syndromes are a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Chromosomal abnormalities and gene mutations have been shown to have essential roles in pathogenesis and correlate with prognosis. Molecular markers, however, are not integrated into currently used prognostic systems. The goal of this study is to identify plasma microRNAs useful for classification and risk stratification of myelodysplastic syndromes. We applied a novel, high-throughput digital quantification technology (NanoString) to profile microRNA expression in plasma samples of 72 patients with myelodysplastic syndromes and 12 healthy individuals. We correlated these results with overall survival. In patients with myelodysplastic syndromes associated with a diploid karyotype, we identified and validated a 7-microRNA signature as an independent predictor of survival with a predictive power of 75% accuracy (P=0.008), better than those of the International Prognostic Scoring Systems and the MD Anderson Prognostic Lower Risk Prognostic Model. We also identified differentially expressed plasma microRNAs in patients with myelodysplastic syndromes versus healthy individuals and between patients with myelodysplastic syndromes associated with different cytogenetic features. These results validate the utility of circulating-microRNA levels as noninvasive biomarkers that can inform the management of patients with myelodysplastic syndromes. Our findings also shed light on interactions of gene regulation pathways that are likely involved in the pathogenesis of myelodysplastic syndromes.

509. Regulated Expression of IL-12 as Gene Therapy Concomitant with Blockade of PD-1 for Treatment of Glioma

The utility of immunotherapy in the treatment of glioma may be improved through combination therapies that enhance cytotoxic immune-activation while concomitantly reducing immunosuppression. We provide data in mice to support evaluation of combining controlled local interleukin 12 (IL-12) administration and blockade of programmed cell death protein 1 (PD-1) in humans. To circumvent challenges surrounding the uncontrolled activation, we have implemented clinical trials using a replication-incompetent adenovirus engineered to conditionally express IL-12 (Ad-RTS-IL-12), via our RheoSwitch Therapeutic System® (RTS®) gene switch. When directly injected intra-tumorally in pre-clinical or clinical studies, IL-12 expression is “off” when devoid of the activator ligand (veledimex, V) and IL-12 production is turned “on” (in a dose-dependent manner) by oral administration of veledimex. PD-1 inhibitors using therapeutic monoclonal antibodies (mAbs) demonstrate an ability to reverse tumor immunosuppression and are effective in the treatment of some cancers. In the present pre-clinical study we assessed the effects of Ad-RTS-mIL-12+veledimex (Ad+V) alone, Ad 5×109 viral particles (vp) + V 10-30mg/m2/day for 14 days or in combination with the antiPD-1-specific mAb RMP1-14 (antiPD-1, 7.5 & 15.0 mg/m2 for 4/day for 5 days i.p.) in the orthotopic GL-261 mouse model. All mice without treatment succumb to disease progression by Day 35. Eighty days after immunotherapy, 70-80% receiving Ad +V monotherapy survived, 30-40% receiving antiPD-1 monotherapy survived and 100% receiving Ad +V 30 mg/m2 + antiPD-1 15.0 mg/m2 combination survived. There was an increase in tumor IL-12 (100 pg/mg) which was 15-times greater than that of plasma peak 5 days after Ad +V. Furthermore, the combination of Ad +V+antiPD-1 sustained peak IL-12 levels in tumor which was associated with a 100-150% increase of activated T cells in the spleen compared with the minimal changes observed with either immunotherapy alone. In addition, there was an additive reduction in regulatory T cells (FOXP3) compared with monotherapies. In summary we demonstrate that controlled local immunostimulation with IL-12 combined with inhibition of PD-1 is an attractive approach for the treatment of glioma. Since both Ad-RTS-IL-12 and mAb blocking PD-1 are clinically available, these data provide impetus for evaluating this combination immunotherapy in humans.

Abstract A193: Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection

Myelosuppressive and ablative regimens are used to deplete lymphocytes in some patients before adoptive cell therapy. CD19-specific engineered T-cells have been used successfully to eliminate B-cells in patients with B-cell leukemias and lymphomas. Prolonged immunosuppression, including B-cell aplasia is associated with increased risk of invasive fungal infection (IFI). Thus, patients will benefit if bio-engineered T cells can control IFI as well as kill tumor cells. The Sleeping Beauty gene transfer system was used to render T cells to express a chimeric antigen receptor (CAR) on the cell surface to redirect specificity via Dectin-1. Upon binding with the β-1,3-glucan sugar moiety found in IFI, Dectin-1 CAR+ T cells signal via chimeric CD28 and CD3-ζ signaling domain. The activated T cells secrete perforin, granzyme and granulysin to damage hyphae and thus inhibit the hyphal growth of Aspergillus and Candida. The T cells also secrete IFN-γ to activate other immune cells to target IFI. To ready the Dectin-1 CAR-modified T cells for the human application, we have modified the Fc domain of the extracellular stalk to alleviate deleterious uptake by Fcγ receptors and thus in vivo loss of infused T cells. We have shown that the Dectin-1 CAR+ T cells do not kill the yeast form of Candida so normal commensals in the gut microbiota should not be affected. We have also demonstrated that Dectin-1 CAR+ T cells can control Aspergillus infection in the presence of immunosuppressive drugs at physiological concentrations. Finally, we have generated dual CAR+ T cells by co-expressing a CD19-specific CAR with Dectin-1 CAR rendering the resultant genetically modified T cells able to target both B-cell malignant cells as well as fungal hyphae. These dual CAR-T cells can thus control both leukemia and invasive fungal infections. These data provide a path forward for clinical trials testing Dectin-1 CAR+ T cells. Citation Format: Pappanaicken R. Kumaresan, Nathaniel Albert, Harjeet Singh, Simon Olivares, Sourindra N. Maiti, Tiejuan Mi, Helen Huls, Richard E. Champlin, Dimitrios P. Kontoyiannis, Laurence J.N. Cooper. Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection . [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A193.