Kappa mAb Research Articles

Changes in immune cell populations following KappaMab, lenalidomide and low-dose dexamethasone treatment in multiple myeloma.

Lenalidomide (LEN) is used to treat multiple myeloma (MM) and shows in vitro synergy with KappaMab (KM), a chimeric antibody specific for Kappa Myeloma antigen, an antigen exclusively expressed on the surface of kappa-restricted MM cells. Lenalidomide, dexamethasone (DEX) and KM control MM via multiple immunomodulatory mechanisms; however, there are several additional effects of the drug combination on immune cells. Lenalidomide can increase T cell and NKT cell cytotoxicity and dendritic cell (DC) activation in vitro. We investigated the immune cell populations in bone marrow of patients treated with KM, LEN and low-dose DEX in kappa-restricted relapsed/refractory MM ex vivo and assessed association of those changes with patient outcome. A cohort (n = 40) of patients with kappa-restricted relapsed/refractory MM, treated with KM, LEN and low-dose DEX, was analysed using a mass cytometry panel that allowed identification of immune cell subsets. Clustering analyses were used to determine significant changes in immune cell populations at time periods after treatment. We found changes in five DC and 17 T-cell populations throughout treatment. We showed an increase in activated conventional DC populations, a decrease in immature/precursor DC populations, a decrease in activated CD4 T cells and an increase in effector-memory CD4 T cells and effector CD8 T cells, indicating an activated immune response. These data characterise the effects of LEN, DEX, and KM treatment on non-target immune cells in MM. Treatment may support destruction of MM cells by both direct action and indirect mechanisms via immune cells.

Validated Method Based on Immunocapture and Liquid Chromatography Coupled to High-Resolution Mass Spectrometry to Eliminate Isatuximab Interference with M-Protein Measurement in Serum

In multiple myeloma (MM) disease, malignant plasma cells produce excessive quantities of a monoclonal immunoglobulin (Ig), known as M-protein. M-protein levels are measured in the serum of patients with MM using electrophoresis techniques to determine the response to treatment. However, therapeutic monoclonal antibodies, such as isatuximab, may confound signals using electrophoresis assays. We developed a robust assay based on immunocapture and liquid chromatography coupled to high-resolution mass spectrometry (IC-HPLC-HRMS) in order to eliminate this interference. Following immunocapture of Ig and free light chains (LC) in serum, heavy chains (HC) and LC were dissociated using dithiothreitol, sorted by liquid chromatography and analyzed using HRMS (Q-Orbitrap). This method allowed the M-proteins to be characterized and the signals from isatuximab and M-proteins to be discriminated. As M-protein is specific to each patient, no standards were available for absolute quantification. We therefore used alemtuzumab (an IgG kappa mAb) as a surrogate analyte for the semiquantification of M-protein in serum. This assay was successfully validated in terms of selectivity/specificity, accuracy/precision, robustness, dilution linearity, and matrix variability from 10.0 to 200 μg/mL in human serum. This method was used for clinical assessment of samples and eliminated potential interference due to isatuximab when monitoring patients with MM.

The clinical application of camrelizumab on advanced hepatocellular carcinoma

ABSTRACT Introduction Camrelizumab (also known as SHR-1210), a humanized monoclonal antibody against PD-1, has been shown to block the binding of PD-1 to PD-L1 and consequently inhibit the immune escape of tumor cells. Recently, camrelizumab was approved as a second-line drug for previously treated advanced hepatocellular carcinoma in China. Areas covered In this paper, the chemical properties, mechanism of action, pharmacokinetics, clinical efficacy, safety, and tolerability of camrelizumab for the treatment of advanced hepatocellular carcinoma are introduced in detail. The strategy for combination therapy and the potential application of camrelizumab in other solid tumors are briefly described. We performed a systematic review of the literature in PubMed and the following keywords were used: ‘SHR-1210,’ ‘Camrelizumab,’ and ‘hepatocellular carcinoma.’ Expert opinion Camrelizumab is a selective, humanized, high-affinity IgG4 kappa mAb against PD-1. Camrelizumab showed promising antitumor activity and manageable toxicities and offers a new second-line drug option for patients with advanced hepatocellular carcinoma. Reactive cutaneous capillary endothelial proliferation is a novel but prevalent immune-related dermatologic toxicity of camrelizumab, which is mild, reversible, and predictable. More clinical trials of camrelizumab are ongoing to develop combination therapy strategies and new indications for malignancies.

Evaluating Isatuximab Interference with Monoclonal Protein Detection By Immuno-Capture and Liquid Chromatography Coupled to High Resolution Mass Spectrometry in the Pivotal Phase 3 Multiple Myeloma Trial, Icaria-MM

Recent advances in multiple myeloma (MM), have seen the advent of several therapeutic monoclonal antibodies (mAb) targeting antigens on malignant plasma cells such as CD38 and SLAMF7. Evaluation of depth of response through quantification of M-Protein, by Serum Protein Electrophoresis (SPEP) and Immuno-Fixation Electrophoresis (IFE) presents a particular challenge for clinical laboratories when therapeutic mAbs are used. Specifically, therapeutic mAbs can confound SPEP and IFE measurements when they overlap with patient serum M-protein. Such interference could lead to an inaccurate determination of depth of response according to International Myeloma Working Group (IMWG) criteria Isatuximab (Isa), an IgG-kappa, anti-CD38 mAb, binds a specific epitope on CD38, which is widely expressed on MM cells. ICARIA-MM was a phase 3, prospective, randomized, open-label, active-controlled, multicenter study comparing isatuximab in combination with pomalidomide plus dexamethasone (Isa-Pd) to pomalidomide plus dexamethasone (Pd) in patients with relapsed/refractory multiple myeloma (RRMM). ICARIA-MM enrolled patients who had received ≥2 prior lines of therapy, including lenalidomide (len) and a proteasome inhibitor (PI). ICARIA-MM demonstrated that Isa-Pd significantly improved PFS, overall response and depth of response with a trend in overall survival benefit and a manageable toxicity profile in these patients. To overcome the potential interference of isatuximab with M-Protein in patients enrolled in ICARIA-MM, we developed and validated a hybrid assay using Immuno-Capture and Liquid Chromatography coupled to High Resolution Mass Spectrometry (IC-LC-HRMS). This method enriches for serum immunoglobulins by immuno-capture. Heavy chains (HC) and light chains (LC) are then dissociated and sorted using Liquid Chromatography. M-Protein and isatuximab LC are analyzed using HRMS which allows to differentiate their monoisotopic intact mass. A small number of patients (24/154; 15.6% in the Isa-Pd arm and 5/153; 3.3% in the Pd arm) fulfilled all criteria for CR (100% M-protein reduction on SPEP, bone marrow <5% plasma cells), but remained IFE positive as per IRC (near-CR category). Serial serum samples from 22 of 24 patients in the Isa-Pd arm were available for MS analysis. Separation of the M-protein and isatuximab signals was performed by IC-LC-HRMS. Using a semi-quantitative approach and applying a sensitivity threshold of 0.25g/dL for IFE-positivity (sensitivity threshold of IFE test per Covance central lab), we identified 11 of 22 patients as having M-protein levels below the threshold for IFE positivity, indicating that the true CR-rate was underestimated in the ICARIA-MM study due to interference. Of the 11 patients with MS M-protein values below the IFE sensitivity threshold, 10 were IgG (8 IgGκ, 2 IgGλ) and one was lambda LC disease with undetectable baseline HC. Baseline myeloma isotype distribution was different in patients with MS M-protein values above IFE sensitivity threshold (4 IgA, 6 IgG, 1 kappa light chain). This confirms that M-protein interference mediated by isatuximab, an IgG kappa mAb, is predominantly seen in patients with IgG isotype (mainly IgGκ), and in LC patients in whom a detectable IgG heavy chain can now be identified. At primary analysis, 4 of 11 patients with MS M-protein values above IFE sensitivity level, versus 2 of 11 patients with MS M-protein values below IFE sensitivity level had progressed. Median PFS was 13.9mo in patients with MS M-protein values above IFE sensitivity level, versus 15.21mo in patients with MS M-protein values below IFE sensitivity level. In conclusion, MS can be successfully used to separate endogenous M-protein from therapeutic isatuximab serum levels. In this subpopulation with excellent response to Isa-Pd, while most patients are still progression-free, a trend can be observed towards longer PFS in patients who would be considered IF negative after MS versus patients remaining IF positive. Disclosures Finn: Sanofi Oncology: Employment. Macé:Sanofi Oncology: Employment. Campana:Sanofi: Employment. Le-Guennec:Sanofi: Employment. Muccio:Sanofi Oncology: Employment. Tavernier:Sanofi Oncology: Employment. Rouchon:Sanofi Oncology: Employment. Roccon:Sanofi Oncology: Employment. Dai:Sanofi Oncology: Employment. Boutet:Sanofi Oncology: Employment. Mouret:Sanofi Oncology: Employment. Pradeilles:Sanofi Oncology: Employment. Hugla:Sanofi Oncology: Employment. Engelvin:Sanofi Oncology: Employment. DiNoto:Sanofi Oncology: Employment. van de Velde:Sanofi: Employment. Fedeli:Sanofi Oncology: Employment. OffLabel Disclosure: Isatuximab is an investigational agent and has not yet been approved by the US Food and Drug administration or any other regulatory agency.

Long-Term Efficacy of Ustekinumab With and Without Concomitant Immunosuppressants for Crohnʼs Disease: Results From IM-UNITI Long-Term Extension Through 2 Years

Introduction: Ustekinumab (UST) is a fully human immunoglobulin G1 kappa mAB to human IL 12/23p40 approved for the treatment of moderate to severe active Crohn's disease (CD). The continuing IM-UNITI long-term extension (LTE) evaluates the efficacy and safety of subcutaneous (SC) UST through approximately 5 years of treatment. Results through maintenance week 44 previously demonstrated no apparent benefit of concomitant immunosuppressant (IMM) use on efficacy, drug levels or immunogenicity of UST. Results through 2 years are reported herein. Methods: 1281 patients entered the maintenance study, including 397 UST induction responders in the primary population (randomized to placebo (PBO) SC; n=133, UST 90mg SC q12w (q12w); n=132, or UST 90mg SC q8w (q8w); n=132). A one-time dose adjustment to UST 90mg SC q8w occurred in randomized patients who met loss of response (LOR) criteria between weeks 8 & 32. All patients completing week 44 were eligible to enter the LTE continuing the treatment they were on at week 44. This analysis included the 82 patients on UST q8w from the primary population who did not meet LOR criteria for dose adjustment and entered the LTE. Clinical remission at each study visit, serum UST concentrations from maintenance weeks 44 to 92, and immunogenicity were assessed in patients taking vs. not taking concomitant IMMs. Results: Baseline use of concomitant IMMs in the randomized UST q8w LTE population was 35.4% (29/82). Patients that were not on IMMs at baseline (64.6%, 53/82), all remained off IMMs through week 92. Rates of remission in the q8w group were not higher among patients with baseline IMM use and were similar through week 92 (Table 1). Furthermore, concomitant use of immunomodulators did not appear to have any notable or consistent effect on serum UST concentrations or antidrug- antibody formation at any timepoint examined (Table 1). Conclusion: Data through 2 years of UST treatment (90mg q8w maintenance) in the IM-UNITI LTE study suggest that concurrent use of immunomodulators does not increase remission efficacy. Further supporting the notion that concomitant use of IMM with UST is not necessary, no effect on serum UST concentrations or antidrug antibodies was seen across all timepoints, in contrast to findings with other biologic agents.573 Figure 1 No Caption available.

95TiP - A Phase 3, Randomised, Double-Blind, Placebo-Controlled, International Study of Medi4736 in Patients with Locally Advanced, Unresectable, Stage 3 Nsclc Who Have not Progressed Following Platinum-Based, Concurrent Chemoradiation Therapy (PACIFIC)

Background: Standard treatment for locally advanced NSCLC has consisted of concurrent radiation with a platinum-based chemotherapy. Nonetheless, the majority of patients ultimately die due to cancer recurrence. Chemotherapy and radiotherapy upregulate the expression of programmed cell death ligand-1 (PD-L1), an inhibitory checkpoint that cancer cells harness to evade antitumour immune responses. MEDI4736 is a human IgG1 kappa mAb that blocks PD-L1 binding to its partner receptor; it is engineered to avoid antibody dependent cell-mediated cytotoxicity.

Response to the combination of cetuximab, bevacizumab and irinotecan (cetux/bev/iri) in patients (pts) previously treated with 3rd-line panitumumab, following disease progression on oxaliplatin, irinotecan and a fluoropyrimidine

14586 Background: There are 2 monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) available for the treatment of advanced metastatic colorectal cancer- panitumumab, a fully human IgG2 kappa mAb and cetuximab, a chimeric IgG1 mAb. The ability to administer one agent after prior treatment with the other is unknown and further, not trivial given the possibility for cross-reactivity. In our institution, we evaluated an exploratory treatment schedule. Methods: We report the status of 8 colon cancer pts treated with panitumumab monotherapy as 3rd-line and subsequently treated with cetux/bev/iri as salvage chemotherapy. All were initially treated on 1 of 2 multicenter phase 2 studies enrolling pts on panitumumab; all pts had documented progressive disease (PD) during or after fluoropyrimidine and adequate doses of irinotecan and oxaliplatin, and low (1–9%) or high (=10%) EGFR tumor membrane staining by IHC. Pts received panitumumab 6 mg/kg q2weeks (wks) until PD or intolerability. Following PD they received bev 5 mg/kg and iri 250 mg/m2 q2wks and cetux 400 mg/m2, then 250 mg/m2 qwk. The endpoint was time to tumor progression (TTP). Results: The 8 pts (2 men, 6 women) were among those enrolled in 1 center. Median age was 67 years (range 51–81); 6 pts had low and 2 pts had high EGFR tumor expression. The median number of cycles of panitumumab was 8 (range 3–14); 1 had a partial remission (PR) for 24 wks, 4 had stable disease (SD) for a median of 14 wks (range 12–16) and 3 pts had PD. Following PD on panitumumab pts received cetux/bev/iri. The median number of cycles was 13 (range 2–17+); 1 had a PR of 20 wks, 6 had SD for a median of 20 wks (range 8–32+) and 1 pt had PD after 4 cycles. Median TTP was 24 wks (range 7–36+). Conclusion: In these 8 pts, prolonged TTP to the combination of cetux/bev/iri was observed following PD on panitumumab and standard chemotherapy. This combination may provide a potential treatment strategy for these heavily pretreated pts with advanced colon cancer. However, further investigation is needed. No significant financial relationships to disclose.

A unique population of circulating autoantibodies promotes central nervous system remyelination.

In previous studies we demonstrated that the humoral immune response directed against unique central nervous system (CNS) antigens enhanced CNS remyelination in the Theiler's virus experimental model of multiple sclerosis (MS). To expand on this observation, a mouse IgM kappa monoclonal antibody (mAb) which enhances CNS remyelination, was raised against normal mouse spinal cord homogenate. Characterization of this mAb revealed that it is polyreactive towards variety of intracellular antigens but also reacts to an unidentified surface antigen on oligodendrocytes. The mAb is encoded by germline immunoglobulin genes without somatic mutations consistent with the observation that it is a natural autoantibody. Recently we generated another mouse IgM kappa mAb raised against normal spinal cord homogenate, which also promotes CNS remyelination. Further characterization revealed that both mAbs which promote remyelination have similar binding characteristics. Conventionally Abs which recognize normal CNS components, especially oligodendrocytes or myelin, have been considered to be a disease marker or be involved in the pathogenesis of MS. However, we have identified a unique population of circulating autoantibodies which are beneficial for myelin repair. Therefore this observation indicates the need to reevaluate autoantibody production against myelin components in CSF and blood as markers of disease activity versus repair in MS.

Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme.

We tested 49 BALB/c antilysozyme mAb from seven intervals during the immune response to lysozyme for patterns of specificity and avidity. We found that the antibody epitopes in composite covered at least 80% of the lysozyme surface, and their patterns of overlap suggest a continuum of potential antibody epitopes. Previously observed regional specificities, which emerged at different times in the immune response, were more discretely defined in late response antibodies, when the majority of mAb could be assigned to one of three functionally nonoverlapping complementation groups. The area covered by each antigenic region may be greater than an individual epitope, and may include multiple epitopes that overlap structurally and functionally to varying degrees. Connectivity between antigenic regions was seen in interactions among early and late stage antibodies, and among secondary stage mAb, but not among tertiary stage mAb from hyperimmunized mice. Patterns of overlap of early and late response antibodies suggest that the organization of antibody specificities change during the progression from primary to secondary to tertiary response. Over the same period in the response, the average relative avidity of IgG1 kappa mAb did not increase, suggesting that "affinity maturation" of serum antibodies reflects an increase in the number and diversity of antibodies, rather than an overall increase in the avidity of individual antibodies.

High level expression on a chimeric anti-ganglioside GD2 antibody: genomic kappa sequences improve expression in COS and CHO cells.

We report a flexible strategy for the high level expression of a recombinant human monoclonal antibody (mAb) in Chinese hamster ovary (CHO) cells, initially using COS monkey kidney cell transfections to evaluate rapidly modifications to immunoglobulin (Ig) DNA constructs. Using sequential transfections with two amplifiable markers, we generated CHO cell lines and clones that secrete 80-110 micrograms/10(6) cells/24 hours of a mouse-human chimeric IgG1 kappa mAb. This cellular productivity is considerably greater than most murine hybridomas and transfected myelomas. Our data also demonstrate that genomic kappa sequences can improve mAb expression in COS and CHO cells. As a paradigm, we focused our expression studies on a human chimeric form of 3F8, a murine mAb that binds to ganglioside GD2 on neuroblastoma and melanoma tumor cells.

Cloning and sequence analysis of the VH and VL regions of an anti-myelin/DNA antibody from a patient with peripheral neuropathy and chronic lymphocytic leukemia.

We have cloned and determined the nucleotide sequence of the Ig VH and VL region genes of an IgM kappa mAb that binds to denatured DNA and myelin from a patient (POP) with chronic lymphocytic leukemia and peripheral neuropathy. Sequence analysis indicates that the V region of the kappa L chain gene (PopVK) has 99% homology to a V kappa IIIa germ-line gene and the V region of the mu H chain gene (PopVH) has 96% homology to the VH26 germ-line gene that is a member of the VH3 gene family. It is likely the V kappa and VH genes arose from these respective germ-line genes via somatic mutation or from closely related genes. V kappa III genes have frequently been used by other IgMk mAb especially those with rheumatoid factor activity, and the VH26 gene with no somatic mutation has been used by several anti-DNA antibodies, suggesting the possibility of preferential association of these or related germ-line genes with autoantibodies. The minor differences between the sequences of POP's VH and V kappa genes and sequences used by other autoantibodies, may be responsible for this antibody's crossreactivity with myelin and, as a result, the autoimmune neuropathy.

Differential regulatory signals delivered by antibody binding to the CD28 (Tp44) molecule during the activation of human T lymphocytes.

Molecule CD28 (Tp44) is expressed on the surface of majority of human T cells and has been implicated to play an active role in the regulation of T cell growth. The present study examines the effect of antibody binding to the CD28 molecule during T cell activation. Anti-CD28 but not isotype-matched anti-CD5 mAb consistently augmented anti-CD3-induced and IL-2-induced T cell proliferation and subsequent release of soluble CD25 molecule. When added together, mAb anti-CD28 and anti-CD5 acted synergistically to cause 2- to 7-fold enhancement of T cell activation induced by anti-CD3 mAb or IL-2 with no effect on the development of non-MHC-restricted IL-2-activated killer T cells. In contrast, alloantigen-induced T cell proliferation, soluble CD25 release, and the subsequent development of CTL were all inhibited by anti-CD28 mAb. Moreover, alloantigen-induced proliferative response of both CD4+ and CD8+ T cells was inhibited by anti-CD28 without affecting the cytolytic effect of CTL. Because valency of anti-CD28 binding has been implicated as an important factor in signal transduction, this was explored in the allogeneic MLR by using Fab and F(ab')2 fragments of anti-CD28 mAb and anti-mouse kappa mAb. The inhibitory effect of anti-CD28 mAb in the MLR was reversed by cross-linking of anti-CD28 mAb with anti-mouse kappa mAb. In addition, cross-linking of the CD28 molecule on alloactivated T lymphoblasts but not that on resting T cells with anti-CD28 and anti-mouse kappa induced their proliferation in the absence of the priming alloantigen. These results indicate that stimulatory or inhibitory signals delivered through the CD28 molecule are determined by the degree of cross-linking of this molecule. In addition, these results also suggest that Ag-induced CD3-TCR-mediated T cell responses are more dependent on signals delivered through the CD28 molecule than those induced with anti-CD3, and thus these results have implications for potential use of anti-CD28 in sustained propagation of Ag-specific T cells.

Primary structure of IgE monoclonal antibodies expressing an intrastrain crossreactive idiotype.

We have obtained amino acid sequences (by mRNA and amino acid sequencing) for two IgE kappa mAb that have specificity for the Ars hapten group and are related to the major idiotypic family, CRIA (crossreactive idiotype A), in the A strain of mouse. One mAb, SE20.2, fully expresses CRIA; the other, SE1.3, possesses some but not all of the characteristic idiotopes. Both IgE proteins contain VH and V kappa segments that are closely related to those associated with CRIA. The D segment of SE20.2 is also typical of CRIA+ mAb, but that of SE1.3 is one amino acid residue longer. Chain recombination experiments indicated that the L chain of SE1.3 is fully capable of supporting CRIA expression. Its deficiency with respect to idiotopes of CRIA was attributed to the extra amino acid in the D region and/or substitutions in the VH segment. A major objective was to ascertain the frequency of somatic mutations in IgE. For the VH segment (amino acids 1-98) of SE20.2, there are only three nucleotide differences and one uncertainty with respect to the nucleotide sequence of the germline gene associated with CRIA. A somewhat higher frequency of substitutions is present in the VH segment of SE1.3. The VK amino acid sequences of the IgE proteins are nearly identical to those of a prototype of the CRIA family, mAb R16.7. The results are discussed with reference to the mechanism of the IgM to IgE switch.

Murine monoclonal antibody LAM2 defines cell membrane determinant with preferential expression on human lung small-cell carcinoma and squamous-cell carcinomas.

Murine monoclonal antibodies (MAbs) were generated against a human undifferentiated lung carcinoma cell line. The hybridoma designated LAM2 produced an IgM kappa MAb with reactivity to the cell membrane. Indirect immunofluorescence staining and radioimmunoassay showed LAM2 antibody to react preferentially with lung small-cell carcinoma (SCC) cell lines and squamous-cell carcinoma (SQC) cell lines. LAM2 antibody also stained primary cultures of normal bronchial epithelial cells, but was unreactive with human erythrocytes and nucleated marrow cells. Indirect immunofluorescent staining with LAM2 antibody was performed on frozen sections of human tumor tissues and normal tissues. LAM2 antibody stained all 8 SCC carcinomas, 4 of 5 SQC of the lung and head and neck region, and 2 or 4 lung large-cell carcinomas. No staining was seen on lung adenocarcinomas, breast carcinomas, ovarian carcinomas, renal cell carcinomas, colon carcinomas, or mesotheliomas. Staining was present on sections of normal bronchus, but not on normal lung parenchyma, liver, kidney, adrenal or skin. While LAM2 antibody was highly reactive with all SCC examined, its antigenic determinant was not expressed in other cell lines and tumors of presumed neuroectodermal origin, including neuroblastoma, melanoma, and bronchial carcinoid. Radioimmunoprecipitation showed the antigen defined by LAM2 antibody to have two major bands of approximate molecular weights of 45,000 and 125,000. The selective reactivity of LAM2 antibody with SCC and SQC, but not with most other tumor tissues and normal tissues, makes it a good candidate for use in clinical diagnosis and possibly serotherapy.