Targeting CD20 Research Articles

Subcutaneous epcoritamab + R-DHAX/C in patients (pts) with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) eligible for autologous stem cell transplant (ASCT): Preliminary phase 1/2 results.

7528 Background: Current standard of care for pts with R/R DLBCL eligible for ASCT includes salvage immunochemotherapy, such as rituximab, dexamethasone, cytarabine, and oxaliplatin or carboplatin (R-DHAX/C), followed by consolidation with high-dose therapy (HDT) and ASCT. However, salvage therapy fails in many pts, and novel treatment (Tx) options are needed to improve response and long-term outcomes. Epcoritamab, a bispecific antibody targeting CD3 on T cells and CD20 on B cells, had a manageable safety profile and meaningful single-agent antitumor activity in pts with heavily pretreated B-cell non-Hodgkin lymphoma (NHL) in the dose-escalation part of the EPCORE NHL-1 trial. Here we present initial results from arm 4 (epcoritamab + R-DHAX/C) of the EPCORE NHL-2 phase 1/2 trial (NCT04663347). Methods: Adults with R/R CD20+ DLBCL who were eligible for HDT-ASCT were enrolled. Pts received standard R-DHAX/C with subcutaneous epcoritamab (QW, 21-d cycle [C] 1–3). If HDT-ASCT was postponed, pts could continue epcoritamab monotherapy (28-d C: QW, C4; Q2W, C5–9; Q4W, C≥10) until disease progression or unacceptable toxicity. Step-up epcoritamab dosing and corticosteroids were required to mitigate CRS in C1. Response was determined by PET-CT per Lugano criteria. Results: As of Jan 26, 2022, 27 pts (median age, 59 y; range, 30–75) had received epcoritamab + R-DHAX/C. Of these, 74% had received 1 prior line of therapy, the remainder (26%) 2–3 prior therapies; 3 pts had prior CAR T. The majority of pts (70%) had primary refractory disease. The most common Tx-emergent adverse events (any grade [G]) were thrombocytopenia (67%), neutropenia (48%; febrile neutropenia, 19% in all pts), infections (37%), nausea (37%), and anemia (33%). Adverse events of special interest were CRS in 8 pts (30%; all G1/2) and ICANS in 1 pt (G2; discontinued epcoritamab, pt with prior CAR T). No clinical tumor lysis syndrome was observed. CRS events occurred early in Tx; median time to resolution was 2 d. Of the 23 evaluable pts, 11 pts underwent ASCT; among these, overall response rate (ORR) was 100% prior to ASCT (82% [9/11] complete metabolic response [CMR]; 18% [2/11] partial metabolic response [PMR]). In 12 pts, HDT-ASCT was postponed/canceled and epcoritamab monotherapy continued; of these, 5 (42%, including 2 with prior CAR T) had CMR and 3 (25%) had PMR. The CMR and PMR rates for the entire evaluable population were 61% (14/23) and 22% (5/23), respectively. Conclusions: Subcutaneous epcoritamab in combination with R-DHAX/C in pts with R/R DLBCL had a manageable safety profile. CRS events were of low grade and resolved with standard management. ORR and CMR rate were high. At the time of data cutoff, 70% of pts had either received ASCT or continued epcoritamab Tx in CMR. Clinical trial information: NCT04663347.

Development of Thermoresponsive Protein Complexes for Targeting CD20 Receptors.

Therapeutics targeting cell receptors can elicit biological responses in situ. However, the ability to dictate when and where these responses occur is a current challenge. Therapeutic proteins can be combined with stimuli-responsive peptides to increase targeting and stimuli responsive behavior. To this end the authors genetically engineered an elastinlike polypeptide (ELP) fusion protein for selective ELPylation. The addition of a charged, foldable region provides these protein subunits with varied thermoresponsive properties from their parent ELPs. These subunits have responsive secondary structures, dependent on pH, indicating the capability to form coiled-coils with a complementary peptide tag. A Rituximab conjugate is generated herein, containing the complementary peptide. Upon mixing of the ELP and Rituximab subunits, the resulting protein complexes can target CD20 receptors on Raji B cells, resulting in at least twofold increase in mean fluorescent intensities. These ELP subunits fold in vitro with the complimentary generated Rituximab conjugate. This work provides the basis for the design of a therapeutic stimuli-responsive biomacromolecule for targeting receptors.

Elotuzumab: no benefit for older patients with newly diagnosed multiple myeloma

Major progress has been made in the treatment of older patients (≥65 years) with newly diagnosed multiple myeloma. To date, continuous lenalidomide-based regimens are considered as standard of care for transplantation-ineligible patients with newly diagnosed multiple myeloma.1 In the past 10 years, immunotherapy was established as a new cornerstone of myeloma therapy, in addition to proteasome inhibitors and immunomodulatory drugs. The incorporation of daratumumab, a monoclonal antibody targeting CD38, with lenalidomide and dexamethasone led to progression-free and overall survival improvement in older patients with newly diagnosed multiple myeloma.

Differential expression of circulating miRNAs after alemtuzumab induction therapy in lung transplantation

Alemtuzumab is a monoclonal antibody targeting CD52, used as induction therapy after lung transplantation (LTx). Its engagement produces a long-lasting immunodepletion; however, the mechanisms driving cell reconstitution are poorly defined. We hypothesized that miRNAs are involved in this process. The expression of a set of miRNAs, cytokines and co-signaling molecules was measured with RT-qPCR and flow cytometry in prospectively collected serum samples of LTx recipients, after alemtuzumab or no induction therapy. Twenty-six LTx recipients who received alemtuzumab and twenty-seven matched LTx recipients without induction therapy were included in the analysis. One year after transplantation four miRNAs were differentially regulated: miR-23b (p = 0.05) miR-146 (p = 0.04), miR-155 (p < 0.001) and miR-486 (p < 0.001). Expression of 3 miRNAs changed within the alemtuzumab group: miR-146 (p < 0.001), miR-155 (p < 0.001) and miR-31 (p < 0.001). Levels of IL-13, IL-4, IFN-γ, BAFF, IL-5, IL-9, IL-17F, IL-17A and IL-22 were different one year after transplantation compared to baseline. In no-induction group, concentration of sCD27, sB7.2 and sPD-L1 increased overtime. Expression of miR-23b, miR-146, miR-486, miR-155 and miR-31 was different in LTx recipients who received alemtuzumab compared to recipients without induction therapy. The observed cytokine pattern suggested proliferation of specific B cell subsets in alemtuzumab group and co-stimulation of T-cells in no-induction group.

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab.

Rituximab, a murine–human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects

目的构建一种靶向CD52的嵌合抗原受体T细胞（CD52 CAR-T），探索其对CD52+白血病的治疗效果。方法应用分子克隆技术构建以CD52为抗原结合区的CD52 scFv、4-1BB为共刺激分子的二代CAR-T细胞。慢病毒载体包装后感染T细胞，制备CD52 CAR-T细胞，通过流式细胞术检测CD52 CAR-T细胞CD4、CD8细胞亚群及其分化状态。通过体外杀伤实验、脱颗粒实验及细胞因子的释放等功能实验，观察CD52 CAR-T细胞对CD52+白血病细胞的特异性细胞毒作用。结果①成功构建了pCDH-CD52 scFv-CD8α-4-1BB-CD3ζ-GFP表达载体，感染人T细胞，获得靶向CD52的CAR-T细胞。②感染CD52 CAR表达载体的第6天，T细胞中表达CD52的细胞可被清除［CD52 CAR-T组CD52+ T细胞为（4.48±4.99）％，Vector-T组为（56.58±19.8）％，P＝0.011］。③CD52 CAR-T的自我杀伤不会影响T细胞的增殖，培养后期CD52 CAR-T组的增殖速度高于Vector-T组。④T细胞表型检测发现CD52 CAR可以促进CAR-T细胞向中心记忆T细胞及效应记忆T细胞分化，减少CAR-T细胞向终末效应细胞分化。⑤CD52 CAR-T细胞与HuT78-19t及MOLT4-19t靶细胞以效靶比1∶1共培养24 h，HuT78-19t细胞可被清除，而对MOLT4-19t细胞无明显杀伤作用［（2.66±1.60）％对（56.66％±5.74）％，P<0.001］。⑥脱颗粒实验显示，HuT78-19t细胞对CD52 CAR-T细胞的激活作用显著高于MOLT4-19t细胞［（57.34±11.25）％对（13.06±4.23）％，P<0.001］。⑦CD52 CAR-T与HuT78-19t细胞共培养48 h上清中细胞因子分泌水平［IFN-γ（3706±226）pg/ml、TNF-α（1732±560）pg/ml］远高于MOLT4-19t细胞组［IFN-γ（1577±846）pg/ml、TNF-α（74±12）pg/ml］（P值均<0.01）。结论该研究成功制备了具有抗白血病作用的CD52 CAR-T细胞，为进一步的靶向CD52的免疫治疗奠定了基础。

Short-Time Effect of Alemtuzumab Induction Therapy on B- and T-cell Subsets After Lung Transplantation

<h3>Purpose</h3> Alemtuzumab is a monoclonal antibody targeting CD52, one of the most predominant antigens on the surface of lymphocytes. It induces cell lysis and leads to a profound immune cell depletion. However, little is known how T- and B-cell repopulate and if there is predominance towards certain subtypes. <h3>Methods</h3> B- and T cell subsets were analyzed from whole blood samples of 25 patients, prospectively collected at time of listing (=baseline) and 6 months post-transplant. Cell subsets were measured by FACS. Patients received alemtuzumab induction followed by low-dose tacrolimus and steroids. <h3>Results</h3> Median age of the cohort was 59 years (51-64) and 36% of patients were female. Underlying diagnoses were COPD (36%), fibrosis (20%), CF (12%), PPH (4%), EAA (12%) and others (16%). B- and T cell counts significantly decreased compared to baseline (p=0.009, p<0.001). Within B cell population, relative frequency of IgM⁺CD27⁻CD38^high, IgM⁺CD27⁺CD38^high and regulatory B cells increased 6 months after Tx (p=0.017, p=0.016, p=0.028, respectively). Within T cell population, CD4⁺ T cells decreased 6 months after Tx (p<0.001), while relative frequency of CD8⁺ T cells was significantly higher (p<0.001). A detailed analysis revealed a shift towards CD4⁺CD57⁺(p=0,002), PD1⁺CD4⁺(p<0.001), CD45RA+CD197-CD4⁺ (p=0.0013), CD45RA⁻CD197⁻CD4⁺ (p<0.001), CD27⁻CD28⁺ (p<0.001), CD27⁺CD28⁺ (p<0.001), PD1⁺CD8⁺ (p<0.001), CD45RA⁺CD197⁻CD8⁺ (p=0.039) and regulatory FoxP3⁺-T cells (p=0.005) at 6 months after Tx. <h3>Conclusion</h3> After alemtuzumab induction therapy, the composition of B and T cell subsets is altered compared to baseline. Expansion of subsets such as PD1 T cells and regulatory B- and T cells suggests an immunomodulatory effect of alemtuzumab on cell repopulation.

P04: DARATUMUMAB PLUS BORTEZOMIB AND DEXAMETHASONE VERSUS BORTEZOMIB AND DEXAMETHASONE ALONE IN PATIENTS WITH PREVIOUSLY TREATED MULTIPLE MYELOMA: OVERALL SURVIVAL RESULTS FROM THE PHASE 3 CASTOR TRIAL

Daratumumab (DARA) is a human IgGκ monoclonal antibody targeting CD38 approved in combination with standard-of-care regimens for pts with newly diagnosed multiple myeloma (NDMM) and as monotherapy and in combination with standard-of-care regimens for pts with relapsed/refractory multiple myeloma (RRMM). In the primary analysis of the phase 3 CASTOR study (median follow-up, 7.4 months), DARA plus bortezomib and dexamethasone (D-Vd) significantly prolonged progression-free survival (PFS) versus bortezomib and dexamethasone (Vd) alone in pts with RRMM, and key secondary endpoints (including time to disease progression, rate of very good partial response or better, overall response rate, and minimal residual disease [MRD]–negativity rate) showed a statistically significant benefit favoring D-Vd. Here, we report final overall survival (OS) and updated MRD-negativity and safety results after ~6 years of follow-up. Methods: Pts with RRMM and ≥1 prior line of therapy were randomized 1:1 to receive D-Vd or Vd. All pts received up to 8 (21-day) cycles of Vd (V 1.3 mg/m2 SC on Days 1, 4, 8, and 11; d 20 mg PO or IV on Days 1, 2, 4, 5, 8, 9, 11, and 12). Pts in the D-Vd group also received DARA (16 mg/kg IV QW in Cycles 1-3, Q3W in Cycles 4-8, and Q4W thereafter until disease progression or unacceptable toxicity). The primary endpoint was PFS; OS was a secondary endpoint. Results: In total, 498 pts were randomized (D-Vd, n=251; Vd, n=247). Median (range) age was 64 (30-88) years; pts had received a median (range) of 2 (1-10) prior lines of therapy. At a median (range) follow-up of 72.6 (0.0-79.8) months, the CASTOR study showed a statistically significant and clinically meaningful improvement in OS with D-Vd versus Vd (hazard ratio [HR], 0.74; 95% confidence interval [CI], 0.59-0.92; P=0.0075 [crossing the prespecified stopping boundary of P=0.0323]), representing a 26% reduction in the risk of death with D-Vd (Figure). Median OS was 49.6 (95% CI, 42.2-62.3) months with D-Vd versus 38.5 (95% CI, 31.2-46.2) months with Vd. Prespecified subgroup analyses showed an OS improvement with D-Vd versus Vd across most subgroups, including pts aged ≥65 years; pts who had received 1 or 2 prior lines of therapy; pts with International Staging System stage III disease, high-risk cytogenetic abnormalities, or prior bortezomib treatment; and pts who were refractory to their last prior line of therapy. The most pronounced OS benefit of D-Vd was seen in pts with 1 prior line of therapy (HR, 0.56; 95% CI, 0.39-0.80). D-Vd achieved significantly higher rates of MRD negativity (10–5) versus Vd (15.1% vs 1.6%; P<0.0001). The most common (≥10%) grade 3/4 treatment-emergent adverse events (TEAEs; D-Vd/Vd) were thrombocytopenia (46.1%/32.9%), anemia (16.0%/16.0%), neutropenia (13.6%/4.6%), lymphopenia (10.3%/2.5%), and pneumonia (10.7%/10.1%). Rates of discontinuation due to TEAEs were low and similar between treatment groups (D-Vd, 10.7%; Vd, 9.3%). No new safety concerns were identified with extended follow-up. Conclusion: Treatment with D-Vd significantly prolonged OS compared with Vd alone. These results, together with the OS results observed with DARA in combination with lenalidomide and dexamethasone in the phase 3 POLLUX study, demonstrate for the first time an OS benefit with DARA-containing regimens in RRMM. The greatest OS benefit of D-Vd was observed in pts with 1 prior line of therapy. Our results support early use of D-Vd to maximize pt benefit.

Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells

BackgroundChimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line.MethodsGiven our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated.ResultsCo-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity.ConclusionsHere, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells.

Exploiting Natural Killer Cell Engagers to Control Pediatric B-cell Precursor Acute Lymphoblastic Leukemia.

Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after αβT/B-depleted haplo-HSCT. The NK cell-resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNγ production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46- and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings.

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for...

CD4 T cell dynamics shape the immune response to combination oncolytic herpes virus and BRAF inhibitor therapy for melanoma

BackgroundCombination herpes simplex virus (HSV) oncolytic virotherapy and BRAF inhibitors (BRAFi) represent promising immunogenic treatments for BRAF mutant melanoma, but an improved understanding of the immunobiology of combinations is needed...

IgG-like Bispecific Antibody CD3×EpCAM Generated by Split Intein Against Colorectal Cancer.

Background: Colorectal cancer is a commonly diagnosed cancer with high mortality worldwide. Postoperative recidivation and metastasis still are the main challenges in clinical treatments. Thus, it is urgent to develop new therapies against colorectal cancer. Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in colorectal cancer cells and strongly associated with cancer development. Bispecific antibody (BsAb) is a kind of promising immunotherapy, which could recognize T cells and cancer cells simultaneously to achieve the anti-tumor effects. Methods: A bispecific antibody targeting EpCAM and CD3 with IgG format was genereated by split intein based on the Bispecific Antibody by Protein Splicing” platform. In vitro, the affinity of CD3×EpCAM BsAb was determined by Biolayer interferometry, its cytotoxicity was detected by LDH release assay, T cell recruitment and activation was detected by Flow Cytometry. In vivo, its pharmacokinetic parameters were detected, and anti-tumor effects were evaluated on the tumor cell xenograft mouse model. Results: The results showed that the CD3×EpCAM BsAb could activate and recruit T cells via binding colorectal cells and T cells, which could lead to more potent cytotoxicity to various colorectal cell lines than its parent EpCAM monoclonal antibody (mAb) in vitro. The CD3×EpCAM BsAb had similar pharmacokinetic parameters with EpCAM mAb and inhibits tumor growth on the SW480 tumor cell xenograft mouse model. Conclusion: The CD3×EpCAM BsAb could be a promising candidate for colorectal cancer treatment.

CD38: A Significant Regulator of Macrophage Function.

Cluster of differentiation 38 (CD38) is a cell surface glycoprotein and multifunctional extracellular enzyme. As a NADase, CD38 produces adenosine through the adenosine energy pathway to cause immunosuppression. As a cell surface receptor, CD38 is necessary for immune cell activation and proliferation. The aggregation and polarization of macrophages are affected by the knockout of CD38. Intracellular NAD+ levels are reduced by nuclear receptor liver X receptor-alpha (LXR) agonists in a CD38-dependent manner, thereby reducing the infection of macrophages. Previous studies suggested that CD38 plays an important role in the regulation of macrophage function. Therefore, as a new marker of macrophages, the effect of CD38 on macrophage proliferation, polarization and function; its possible mechanism; the relationship between the expression level of CD38 on macrophage surfaces and disease diagnosis, treatment, etc; and the role of targeting CD38 in macrophage-related diseases are reviewed in this paper to provide a theoretical basis for a comprehensive understanding of the relationship between CD38 and macrophages.

Abstract P2-07-09: Characterization of the tumor microenvironment in patients with hormone receptor positive, HER2 negative early breast cancer

Abstract Background: Patients (pts) with early stage HER2+ breast cancer (HER2+ EBC) are often treated with neoadjuvant chemotherapy (NACT). Recent studies have demonstrated that increased densities of tumor infiltrating lymphocytes (TIL) are associated with increased rates of pathologic complete response (pCR) in HER2+ EBC. However, outside of standard TIL analysis, a more comprehensive description of the tumor microenvironment (TME) and its impact on clinical outcomes in HER+ EBC is not well defined. Using novel methods, we sought to characterize the TME of patients with hormone receptor positive (HR+), HER2+ EBC treated with NACT. In addition to standard H&amp;E based TIL analysis, we utilized quantitative multiplex immunofluorescence (qmIF), spatial analysis and analysis of RNA-based gene signatures to assess for pretreatment TME characteristics associated with pCR to NACT in HER2+/HR+ EBC. Methods: We identified 29 pts with HER2+/HR+ EBC treated with NACT between 2005 and 2015 for which pretreatment biopsy tissue was available. 13 pts (45%) were treated with an anthracycline, 26 (90%) were treated with trastuzumab in addition to chemotherapy and 14 (48%) were treated with trastuzumab and pertuzumab. At surgery, 13 pts (45%) achieved a pCR. A breast cancer pathologist performed TIL analysis according to international guidelines on pretreatment H&amp;E slides. qmIF was performed on 28 pretreatment specimens using an antibody panel targeting CD3, CD8, CD68, FOXP3, and pancytokeratin. We analyzed multispectral images using HALO software to perform tissue segmentation (tumor vs stroma) and phenotypic analysis based on antibody staining patterns. Upon spatially resolving these components, we then performed proximity analysis. We obtained mRNA-based gene signatures via the Nanostring nCounter Breast Cancer 360 Panel for 28 patients. T-tests were performed to evaluate for associations of TME features with pCR. Results: We evaluated the association of pCR with: manual H&amp;E-based TIL assessments, lymphocyte densities and interactions (assessed by spatial proximity) from qmIF, and RNA signatures assessed from the NanoString BC360 panel. Univariate analysis revealed strong associations of inflammation signatures derived from qmIF with pCR, including higher stromal densities of CD3+ (p&amp;lt;0.01), CD8+ (p=0.02) and CD3+CD8-FOXP3- (p=0.02) cells. pCR was also associated with higher ratios of CD3+:CD68+ (p&amp;lt;0.01). This finding remained significant in T-cell subsets CD8+:CD68+ (p=0.04) and was driven by CD3+CD8-FOXP3-:CD68+ (p=0.02). High numbers of lymphocyte interactions, assessed by counting pairs of cells within 30 microns of each other, were also associated with pCR, including CD3+:FOXP3+ (p&amp;lt;0.01) and CD3+CD8-FOXP3-:FOXP3+ (p&amp;lt;0.01). We found no significant associations between pCR and manual H&amp;E based TIL assessments or RNA signatures. Conclusion: In this population of HER2+/HR+ EBC treated with NACT, stromal and intratumoral TIL assessment by standard H&amp;E was not associated with pCR. Higher stromal densities of CD3+, CD8+, and CD3+CD8-FOXP3- TIL subsets were associated with pCR by qmIF and further analysis revealed the presence and proximity of lymphocyte subpopulations correlated with pCR, suggesting that spatial relationships between these populations within the TME contributes to sensitivity to NACT. The lack of association between TILs and pCR in our cohort by standard H&amp;E is likely due to small numbers; however, significance was seen by qmIF, indicating this method as a potentially more sensitive approach to this analysis. Analysis of RNA signatures by NanoString BC360 panel did not reveal signatures associated with pCR . While our study demonstrates the feasibility of this approach to TME evaluation, further studies of larger cohorts are needed to validate markers predictive of response to NACT. Citation Format: Matthew Ryan Kearney, Rami Vanguri, Qi Wang, Kathleen Fenn, Hua Guo, Douglas Marks, Hanina Hibshoosh, Kevin M Kalinsky, Eileen Connolly. Characterization of the tumor microenvironment in patients with hormone receptor positive, HER2 negative early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-09.

Increased Activity of a NK-Specific CAR-NK Framework Targeting CD3 and CD5 for T-Cell Leukemias.

Simple SummaryChimeric antigen receptors (CAR) can redirect the activity of NK cells to target T-cell malignancies. Our results identify that recognition of CD5 molecules in malignant T cells by the CAR leads to improved antitumor response compared to targeting CD3, due to strong downregulation of the CD3 antigen after CD3-CAR treatment. We have also identified that a specific CAR-NK framework has superior activity than a CAR-T framework on NK effector cells.NK effector cells expressing a CAR construct may be used to target T-lineage markers. In this work, we compared the activity of a NK-specific CAR-NK and a CAR-T framework when expressed on NK effector cells to target CD3 and CD5 in T-cell malignancies. Our results show that CD3-CAR-T is more active than CD5-CAR-T to eliminate malignant T cells in vitro, however, CD3-CAR-T were less efficient to eliminate tumor cells in vivo, while CD5-CAR-T had antitumor activity in a diffuse xenograft model. Lack of in vivo efficacy correlated with downregulation of CD3 levels in target T cells after coculture with CD3-CAR effector cells. The CAR-NK framework greatly improved the efficacy of CARs leading to increased degranulation, cytokine secretion and elimination of the tumor xenograft by CD5-CAR-NK effector cells. Finally, all CAR constructs were similarly effective to eliminate malignant T cells in vitro. Our results show that the NK-CAR framework improves the activity of CARs in NK cells and that CD5 would be a better target than CD3 for T-cell malignancies, as dynamic downregulation of target expression may affect in vivo efficacy.

Daratumumab: A review of current indications and future directions

Daratumumab, a human IgG1 kappa monoclonal antibody targeting CD38 has transformed the treatment paradigm of multiple myeloma (MM). With the identification of CD38 as a crucial receptor involved in immune system function, it became an ideal target for monoclonal antibody (mAb) drug development in MM. Daratumumab's unique multifaceted mechanism of action has led to great success in the treatment of relapsed refractory multiple myeloma (RRMM) as well as newly diagnosed multiple myeloma (NDMM) patients. Along with its efficacy comes a low toxicity profile, improved further with the introduction of subcutaneous daratumumab. With such success within MM, daratumumab is now being explored in other disease states. This article will review daratumumab's drug development, practical use, and future potential indications.

Daratumumab combined with dexamethasone and lenalidomide or bortezomib in relapsed/refractory multiple myeloma (RRMM) patients: Report from the multiple myeloma GIMEMA Lazio group.

The multiple myeloma (MM) treatment has changed over the last years due to the introduction of novel drugs. Despite improvements in the MM outcome, MM remains an incurable disease. Daratumumab is a human IgGK monoclonal antibody targeting CD38 with tumor activity associated with immunomodulatory mechanism. In combination with standard of care regimens, including bortezomib (Vd) or lenalidomide (Rd), daratumumab prolonged progression‐free survival (PFS) in patients (pts) with relapsed/refractory multiple myeloma (RRMM) and in new diagnosis MM. We report the data of the MM GIMEMA Lazio group in 171 heavily treated pts who received daratumumab, lenalidomide and dexamethasone (DRd) or daratumumab, velcade and dexamethasone (DVd). The overall response rate was 80%, and the overall survival (OS) and PFS were 84% and 77%, respectively. In addition, pts treated with DRd showed a better median PFS compared to pts treated with DVd, at 12 and 24 months, respectively. The most common hematologic treatment‐emergent adverse events (TAEs) were neutropenia, thrombocytopenia, and anemia. The most common nonhematologic TAEs were peripheral sensory neuropathy and infections. Our data confirmed that DRd or DVd therapy is effective and safe in RRMM pts, and our real‐life analysis could support the physicians regarding the choice of optimal therapy in this setting of pts.

Targeting CD33 for acute myeloid leukemia therapy

BackgroundThe aim of this study was to analyze the level of CD33 expression in patients with newly diagnosed AML and determine its correlation with clinical characteristics.MethodsSamples were collected for analysis from AML patients at diagnosis. We evaluated the level of CD33 expression by flow cytometry analysis of bone marrow. Chi-square or t- tests were used to assess the association between the high and low CD33 expression groups. Survival curves were generated by the Kaplan-Meier and Cox regression model method.ResultsIn this study we evaluated the level of CD33 expression in de novo patients diagnosed from November 2013 until January 2019. The mean value of 73.4% was used as the cutoff for the two groups. Statistical analysis revealed that 53 of the 86 (61.2%) AML patients were above the mean. Although there was no statistical significance between CD33 expression level and gene mutation, FLT3 mutation (P = 0.002) and NPM1 mutation (P = 0.001) were more likely to be seen in the high CD33 group. The overall survival (OS) was worse in the high CD33 group (39.0 m vs. 16.7 m, x2 = 13.06, P < 0.001). The Cox survival regression display that the CD33 is independent prognostic marker (HR =0.233,p = 0.008). Univariate analysis showed that the high expression of CD33 was an unfavorable prognostic factor. Of the 86 patients, CD33-high was closely related to the patients with normal karyotype (x2 = 4.891,P = 0.027), high white blood cell count (WBC, t = 2.804, P = 0.007), and a high ratio of primitive cells (t = 2.851, P = 0.005).ConclusionsThese findings provide a strong rationale for targeting CD33 in combination with chemotherapy, which can be considered a promising therapeutic strategy for AML.