Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Genetic defects of gamma-secretase genes in a multiple myeloma patient with high and dysregulated BCMA surface density: A case report.

Multiple myeloma (MM) cells from 1 out of 20 patient expressed high basal levels of membrane B-cell maturation antigen (BCMA, TNFRSF17, CD269), which was not upregulated by gamma-secretase inhibitor, suggesting a defective BCMA shedding by gamma-secretase. Genetic analyses of the patient's bone marrow DNA showed no mutations within the BCMA coding region, but rather partial deletion of PSEN1 and amplification of PSEN2, which encode alternative catalytic units of gamma-secretase. Altogether the data suggest that pt#12 MM cells express high and dysregulated BCMA with no shedding, due to genetic alterations of one or more gamma-secretase subunits.

BL-01, an Fc-bearing, tetravalent CD20 × CD5 bispecific antibody, redirects multiple immune cells to kill tumors in vitro and in vivo.

BACKGROUND AIMS: The authors describe here a novel therapeutic strategy combining a bispecific antibody (bsAb) with cytokine-induced killer (CIK) cells. METHODS: The authors have designed, produced and purified a novel tetravalent IgG1-like CD20 × CD5 bsAb called BL-01. The bsAb is composed of a fused heavy chain and two free light chains that pair correctly to the heavy chain sequences thanks to complementary mutations in the monoclonal antibody 2 CH1/CL sequences. RESULTS: The authors show that BL-01 can bind specifically to CD20 and CD5 with an affinity of 4-6 nM, demonstrating correct pairing of two light chains to the fused heavy chain. The CD20 × CD5 BL-01 bsAb has a functional human IgG1 Fc and can induce up to 65% complement-dependent cytotoxicity of a CD20+ lymphoma cell line in the presence of human complement, similar to anti-CD20 rituximab. The bsAb also induces significant natural killer cell activation and antibody-dependent cytotoxicity of up to 25% as well as up to 65% phagocytosis by human macrophages in the presence of CD20+ tumor cells. The BL-01 bsAb binds to CD20 and CD5 simultaneously and can redirect CIK cells in vitro to kill CD20+ targets, increasing the cytotoxicity of CIK cells by about 3-fold. The authors finally show that the CD20 × CD5 BL-01 bsAb synergizes with CIK cells in vivo in controlling tumor growth and prolonging survival of nonobese diabetic/severe combined immunodeficiency mice inoculated with a patient-derived, aggressive diffuse large B-cell lymphoma xenograft. CONCLUSIONS: The authors suggest that the efficacy of bsAb in vivo is due to the combined activation of innate immunity by Fc and redirection of CIK cells to kill the tumor target.

Identification of Human SARS-CoV-2 Monoclonal Antibodies from Convalescent Patients Using EBV Immortalization.

We report the isolation of two human IgG1k monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike protein. These mAbs were isolated from two donors who had recovered from COVID-19 infection during the first pandemic peak in the Lombardy region of Italy, the first European and initially most affected region in March 2020. We used the method of EBV immortalization of purified memory B cells and supernatant screening with a spike S1/2 assay for mAb isolation. This method allowed rapid isolation of clones, with one donor showing about 7% of clones positive against spike protein, whereas the other donor did not produce positive clones out of 91 tested. RNA was extracted from positive clones 39-47 days post-EBV infection, allowing VH and VL sequencing. The same clones were sequenced again after a further 100 days in culture, showing that no mutation had taken place during in vitro expansion. The B cell clones could be expanded in culture for more than 4 months after EBV immortalization and secreted the antibodies stably during that time, allowing to purify mg quantities of each mAb for functional assays without generating recombinant proteins. Unfortunately, neither mAb had significant neutralizing activity in a virus infection assay with several different SARS-CoV-2 isolates. The antibody sequences are made freely available.

Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation.

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab')2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.

Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies.

Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo.

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.

Direct involvement of CD56 in cytokine-induced killer-mediated lysis of CD56+ hematopoietic target cells.

Cytokine-induced killer (CIK) cells are in-vitro-expanded T lymphocytes that represent a heterogeneous population. A large majority of CIK cells are CD3(+)CD56(+), and this population has been shown to confer a cytotoxic effect against tumor targets. The scope of this work was to study whether CD56 has a direct role in CIK-mediated cytotoxicity. Blocking of CD56 with the anti-CD56 monoclonal antibody GPR165 significantly reduced CIK-mediated lysis of three CD56(+) hematopoietic tumor cell lines (AML-NS8, NB4, and KCL22), whereas no effect was observed on three CD56(-) hematopoietic tumor cell lines (K562, REH, and MOLT-4). Knockdown of CD56 in CIK cells by short interfering RNA made the cells less cytotoxic against a CD56(+) target, and knockdown of CD56 in target cells with lentiviral short hairpin RNA significantly altered their susceptibility to CIK-mediated lysis. Our data suggest that homophilic interaction between CD56 molecules may occur in tumor-cell recognition, leading to CIK-mediated cell death.

Transcription of Satellite III non-coding RNAs is a general stress response in human cells.

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 10(4)-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions.

Structural and functional characterization of noncoding repetitive RNAs transcribed in stressed human cells.

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs.

Regulated compartmentalization of the putative DEAD-box helicase MDDX28 within the mitochondria in COS-1 cells.

We recently cloned a putative DEAD-box helicase MDDX28 and found that it was localized to the nuclei and mitochondria of COS-1 cells. The mitochondrial localization of MDDX28 is largely diffuse. We have, however, used immunofluorescence and immunogold cytochemistry to show that the MDDX28 protein is localized in a distinct mitochondrial subcompartment in 5-10% of COS-1 cells. This proportion increases to approximately 35% after treatment with ethidium bromide, suggesting upregulation following transcription inhibition. To our knowledge, this is the first example of protein relocation in the mitochondria caused by transcription inhibition. The mitochondrial subcompartmentation of MDDX28 was negatively affected by mutations in a RNA-binding domain and three basic domains previously shown to be important in transcription-dependent intranuclear localization. Furthermore, immunogold cytochemistry and fractionation of rat liver indicated that the protein is a part of an RNA-protein (RNP) complex interacting peripherally with the mitochondrial inner membrane. Our results reveal new principles for regulation of protein localization in the mitochondria and suggest parallels between the function of the MDDX28 protein in the nucleus and mitochondria.

RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies.

Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first alpha-helix of this domain, but not in the beta-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding domain of the protein as the major determinant of this interaction.

Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock.

Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover, heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts. In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli.

Transport signals and transcription-dependent nuclear localization of the putative DEAD-box helicase MDDX28.

The human protein MDDX28 is a putative RNA helicase and a nucleocytoplasmic shuttling protein also localized to the mitochondria. Its localization is novel among RNA helicases. We have studied its intracellular targeting signals and show that the first 20 amino acids of MDDX28 are necessary and sufficient for both mitochondrial import and nuclear export of the protein. Mutation of the five leucines in the sequence to alanines abolished the mitochondrial targeting signal as well as greatly reducing the nuclear export signal, indicating that these signal sequences are highly overlapping. Two short stretches of basic amino acids separated by 44 residues were both necessary and sufficient for full nuclear localization. However, they were not absolutely essential, because the protein was present in 7% of the nuclei when both signals were mutated. This indicates that MDDX28 contains another unidentified weak nuclear localization signal(s). Three basic domains in the N-terminal half of the protein and its RNA binding ability were essential for nucleolar localization as well as transcription-inhibition-dependent localization to nuclear subcompartments. Two of these basic domains were the same as those constituting the nuclear localization signal, suggesting that they are responsible for bringing the protein into the nucleus to the sites of RNA binding. Our results indicate that MDDX28 nucleo-cytoplasmic shuttling is dependent on the availability of nascent RNA.