Key Points PF-06747143, a novel CXCR4 antagonist IgG1 Ab, mobilizes malignant cells from the BM and induces their death via Fc-effector function. PF-06747143 reduces tumor burden in NHL, AML, and MM models, both as a monotherapy or in combination with standard-of-care agents.

Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.

Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C - X - C motif chemokine r eceptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer “homing” to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector–reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4 + cancer cells in solid tumours, but showed limited toxicity to normal CXCR4 + tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression.

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An important aspect of the drug development process is prediction of efficacious and toxic side effects. Profiling of mRNA expression is a powerful approach to analyze the molecular phenotype of cells under various conditions, for example, in response to stimulation by compounds. We attempt to explore the approach of using expression profiling to identify patterns or fingerprints that are correlated with specific drug properties or behaviors. Identification of such expression patterns may also lead to revelation of the potential action mechanism of drugs and fingerprints indicative of certain drug efficacy or side effects. We describe here a strategy that was used to identify a set of genes whose differential expression pattern correlates with activation mode and target specificity of a specific group of drug compounds.

CXCR4 is a chemokine receptor that belongs to the G-coupled protein receptor (GPCR) family. It is over-expressed in various cancers, including solid tumors and hematological malignancies, and correlates with poor prognosis. CXCR4 expressing cells actively respond to CXCL12 (SDF-1), a chemokine constitutively secreted by stromal cells in bone marrow. Activation of CXCR4 induces cell trafficking and homing to the marrow microenvironment, where CXCL12 retains these cells in close contact with marrow stromal cells that provide growth signals, promote self-renewal, and contribute to drug resistance, leading to poor prognosis and relapse. Here we describe the generation of a highly potent and selective anti-CXCR4 humanized IgG1 antagonist Ab (PF-06747143) that binds to human CXCR4 with high affinity and blocks SDF-1-induced Calcium flux and cAMP signaling. We have also characterized the ability of PF-06747143 to induce cell death through three different mechanisms: a) mobilization of cells from CXCL12-rich niches, making them more sensitive to chemotherapy b) direct cell-death through a mechanism dependent on the antibody’s bivalency; c) ADCC- and CDC-dependent cell death through the Fc-region in IgG1 backbone, when in the presence of effector cells or serum proteins. Weekly administration of PF-06747143 at 10 mg/kg, as a monotherapy, significantly improved survival, induced sustained regression and reduced bone marrow tumor burden in various patient population relevant murine disseminated tumor models of Acute Myeloid Leukemia (MV4-11, PDXs), Non Hodgkin Lymphoma (Raji and Ramos), Chronic Lymphocytic Leukemia (JVM-13) and Multiple Myeloma (OPM-2). The CXCR4 IgG1 antibody was also shown to be similar or more efficacious than approved standards of care agents currently employed for treatment of hematological malignancies. The safety and PK/PD profile of PF-06747143 were evaluated in a Non-Human Primate (NHP) exploratory toxicology study. Results from this study indicate that the CXCR4 IgG1 Ab was well tolerated in a two-week exploratory study at pharmacologically relevant doses. Upon treatment with PF-06747143, egression of white blood cells (WBC) from bone marrow (leukocytosis) was noted, which is consistent with target (CXCR4) modulation. Following the peak of leukocytosis between 1-6 hours post antibody administration, the number of circulating WBCs rapidly decreased back to baseline levels at 24 hrs. These results are likely explained by the direct cell killing through the effector function of this IgG1 CXCR4 antibody. Altogether, the promising preclinical efficacy and safety data support clinical evaluation of PF-06747143 in hematological malignacies. Disclosures Pernasetti: Pfizer: Employment. Liu:Pfizer: Employment. Hallin:Pfizer: Employment. Gu:Pfizer: Employment. Ho:Pfizer: Employment. Zhang:Pfizer: Employment. Pascual:Pfizer: Employment. Simmons:Pfizer: Employment. Yan:Pfizer: Employment. Huser:Pfizer: Employment. Wang:Pfizer: Employment. Lam:Pfizer: Employment. Spilker:Pfizer: Employment. Blasi:Pfizer: Employment. Tran:Pfizer: Employment. Kudaravalli:Pfizer: Employment. Ma:Pfizer: Employment. Chin:Pfizer: Employment. Shelton:Pfizer: Employment. Smeal:Pfizer: Employment. Fantin:Pfizer: Employment.

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal quality from the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 µg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 µg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.