A mouse model for evaluation of efficacy and concomitant toxicity of anti-human CXCR4 therapeutics

Abstract

The development of therapeutic monoclonal antibodies through mouse immunization often originates drug candidates that are not cross-reactive to the mouse ortholog. In such cases, and particularly in oncology, drug efficacy studies are performed on human tumor xenografts or with “surrogate” anti-mouse ortholog antibodies if targeting tumor host cells. Safety assessment of drug candidate(s) is performed at a later development stage in healthy non-human primates. While the latter remains necessary before a drug advances into human subjects, it precludes evaluation of safety in disease conditions and drug de-risking during early development. Therefore, mouse models that allow concomitant evaluation of drug efficacy and safety are highly desirable. The C-X-C motif chemokine receptor 4 (CXCR4) is an attractive target for tumor-targeted and immuno-oncology therapeutics, with multiple mouse immunization-derived antibodies undergoing clinical trials. Given the pleiotropic role of CXCR4 in cancer biology, we anticipate continuous interest in this target, particularly in the testing of therapeutic combinations for immuno-oncology. Here, we describe the generation and validation of the first mouse knock-in of the whole coding region of human CXCR4. Homozygous human CXCR4 knock-in (hereafter designated as HuCXCR4KI) mice were viable and outwardly healthy, reproduced normally and nursed their young. The expression pattern of human CXCR4 in this model was similar to that of CXCR4 expression in normal human tissues. The human CXCR4 knock-in gene was expressed as a biologically active protein, thereby allowing normal animal development and adequate”homing” of leukocytes to the bone marrow. To further validate our model, we used an in vivo functional assay of leukocyte mobilization from bone marrow to peripheral blood by blocking CXCR4 signaling. Both an anti-human CXCR4 -specific blocking antibody and the small molecule CXCR4 inhibitor AMD3100 induced increased leukocyte counts in peripheral blood, whereas an anti-mouse CXCR4 –specific blocking antibody had no effect. This new mouse model is useful to evaluate efficacy and safety of anti-human CXCR4 -specific drugs as single agents or in combination therapies, particularly in the oncology, immuno-oncology, wound healing and chronic inflammation therapeutic areas.

Targeting CXCR4 in AML and ALL

“…Their optimal ADC, ADC 713, was a low-affinity humanized IgG1 antibody with reduced Fc-mediator effector function. Tolerability and safety studies performed in a human CXCR4 knock-in mouse model ( 127 ) revealed significant decreases in the numbers of circulating neutrophils and monocytes 3 days after the third injection of ADC 713. Although the numbers of bone marrow lineage-negative/Sca-positive/Kit-positive (LSK) cells and HSPCs were unchanged, there was a significant decrease in granulocyte-monocyte progenitors, suggesting some hematopoietic toxicity.…”

Optimal design, anti-tumour efficacy and tolerability of anti-CXCR4 antibody drug conjugates

“…6c). In our orthotopic in vivo models, mouse CXCL12 activates human CXCR4 8,12 and most MM cells are found in the bone marrow. The serum CXCL12 levels in tumour-naïve NSG mice average those of wild type, tumour-naïve Balb/c mice (Supplementary Fig.…”

“…Besides haematopoietic progenitors, CXCR4 is expressed in leukocytes and other normal adult quiescent tissues (kidney tubular epithelium and adrenal gland) 6,8,42,43 . We reasoned that a payload class whose mechanism of action (MoA) involves blocking cell division would enhance the TI of an anti-CXCR4 ADC.…”

“…6a and Supplementary Table 7). In this mouse model, the coding region of mouse CXCR4 gene has been replaced by the human ortholog sequence and the mice develop an overtly normal haematopoietic system 8 . Thus, HuCXCR4KI mice allow the evaluation of normal tissue toxicity of anti-human CXCR4-specific ADCs.…”

“…As such, global knockouts of CXCR4 and its ligand CXCL12 are embryonic lethal 3–5 . In adult tissues, CXCR4 is expressed in haematopoietic cells, adrenal gland, and kidney tubules 6–8 , whereas CXCL12 is a homeostatic chemokine, being expressed by mesenchymal stromal cells in many tissues 9 . CXCL12/CXCR4 signalling has multiple functions in haematopoietic progenitor cells: maintenance of quiescence, retention in bone marrow and protection from oxidative stress 10–13 .…”