scholarly journals Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 576-585 ◽  
Author(s):  
ML Grossbard ◽  
AS Freedman ◽  
J Ritz ◽  
F Coral ◽  
VS Goldmacher ◽  
...  
Keyword(s):  
Phase I ◽  
B Cell ◽  
Maximum Tolerated Dose ◽  
In Vitro Cytotoxicity ◽  
Bolus Infusion ◽  
Protein Toxin ◽  
Cytotoxicity Studies ◽  
Ricin A

Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin “blocked ricin.” The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.

scholarly journals Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 576-585 ◽  
Author(s):  
ML Grossbard ◽  
AS Freedman ◽  
J Ritz ◽  
F Coral ◽  
VS Goldmacher ◽  
...  
Keyword(s):  
Phase I ◽  
B Cell ◽  
Maximum Tolerated Dose ◽  
In Vitro Cytotoxicity ◽  
Bolus Infusion ◽  
Protein Toxin ◽  
Cytotoxicity Studies ◽  
Ricin A

Abstract Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin “blocked ricin.” The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.

Intrathecal Mafosfamide: A Preclinical Pharmacology and Phase I Trial

2005 ◽  
Vol 23 (7) ◽  
pp. 1555-1563 ◽  
Author(s):  
Susan M. Blaney ◽  
Frank M. Balis ◽  
Stacey Berg ◽  
Carola A.S. Arndt ◽  
Richard Heideman ◽  
...  
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
Phase I Trial ◽  
Neoplastic Meningitis ◽  
In Vitro Cytotoxicity ◽  
Pharmacokinetic Studies ◽  
Preclinical Studies ◽  
Phase Ii Trials ◽  
Cytotoxicity Studies

Purpose Preclinical studies of mafosfamide, a preactivated cyclophosphamide analog, were performed to define a tolerable and potentially active target concentration for intrathecal (IT) administration. A phase I and pharmacokinetic study of IT mafosfamide was performed to determine a dose for subsequent phase II trials. Patients and Methods In vitro cytotoxicity studies were performed in MCF-7, Molt-4, and rhabdomyosarcoma cell lines. Feasibility and pharmacokinetic studies were performed in nonhuman primates. These preclinical studies were followed by a phase I trial in patients with neoplastic meningitis. There were five dose levels ranging from 1 mg to 6.5 mg. Serial CSF samples were obtained for pharmacokinetic studies in a subset of patients with Ommaya reservoirs. Results The cytotoxic target exposure for mafosfamide was 10 μmol/L. Preclinical studies demonstrated that this concentration could be easily achieved in ventricular CSF after intraventricular dosing. In the phase I clinical trial, headache was the dose-limiting toxicity. Headache was ameliorated at 5 mg by prolonging the infusion rate to 20 minutes, but dose-limiting headache occurred at 6.5 mg dose with prolonged infusion. Ventricular CSF mafosfamide concentrations at 5 mg exceeded target cytotoxic concentrations after an intraventricular dose, but lumbar CSF concentrations 2 hours after the dose were less than 10 μmol/L. Therefore, a strategy to alternate dosing between the intralumbar and intraventricular routes was tested. Seven of 30 registrants who were assessable for response had a partial response, and six had stable disease. Conclusion The recommended phase II dose for IT mafosfamide, administered without concomitant analgesia, is 5 mg over 20 minutes.

Polymeric/Inorganic Multifunctional Nanoparticles for Simultaneous Drug Delivery and Visualization

2010 ◽  
Vol 1257 ◽  
Author(s):  
Andrea Fornara ◽  
Alberto Recalenda ◽  
Jian Qin ◽  
Abhilash Sugunan ◽  
Fei Ye ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Contrast Agents ◽  
Gold Nanorods ◽  
In Vitro Cytotoxicity ◽  
In Vivo Studies ◽  
Multifunctional Nanoparticles ◽  
Cytotoxicity Studies

AbstractNanoparticles consisting of different biocompatible materials are attracting a lot of interest in the biomedical area as useful tools for drug delivery, photo-therapy and contrast enhancement agents in MRI, fluorescence and confocal microscopy. This work mainly focuses on the synthesis of polymeric/inorganic multifunctional nanoparticles (PIMN) based on biocompatible di-block copolymer poly(L,L-lactide-co-ethylene glycol) (PLLA-PEG) via an emulsion-evaporation method. Besides containing a hydrophobic drug (Indomethacin), these polymeric nanoparticles incorporate different visualization agents such as superparamagnetic iron oxide nanoparticles (SPION) and fluorescent Quantum Dots (QDs) that are used as contrast agents for Magnetic Resonance Imaging (MRI) and fluorescence microscopy together. Gold Nanorods are also incorporated in such nanostructures to allow simultaneous visualization and photodynamic therapy. MRI studies are performed with different loading of SPION into PIMN, showing an enhancement in T2 contrast superior to commercial contrast agents. Core-shell QDs absorption and emission spectra are recorded before and after their loading into PIMN. With these polymeric/inorganic multifunctional nanoparticles, both MRI visualization and confocal fluorescence microscopy studies can be performed. Gold nanorods are also synthesized and incorporated into PIMN without changing their longitudinal absorption peak usable for lased excitation and phototherapy. In-vitro cytotoxicity studies have also been performed to confirm the low cytotoxicity of PIMN for further in-vivo studies.

scholarly journals First-in-human phase I trial of anti-hepatocyte growth factor antibody (YYB101) in refractory solid tumor patients

2020 ◽  
Vol 12 ◽  
pp. 175883592092679
Author(s):  
Seung Tae Kim ◽  
Jung Yong Hong ◽  
Se Hoon Park ◽  
Joon Oh Park ◽  
Young Whan Park ◽  
...  
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Phase I ◽  
Cancer Patients ◽  
Phase I Trial ◽  
Maximum Tolerated Dose ◽  
Safety And Efficacy ◽  
Hepatocyte Growth

Background: YYB101, a humanized monoclonal antibody against hepatocyte growth factor (HGF), has shown safety and efficacy in vitro and in vivo. This is a first-in-human trial of this antibody. Materials and Methods: YYB101 was administered intravenously to refractory cancer patients once every 4 weeks for 1 month, and then once every 2 weeks until disease progression or intolerable toxicity, at doses of 0.3, 1, 3, 5, 10, 20, 30 mg/kg, according to a 3+3 dose escalation design. Maximum tolerated dose, safety, pharmacokinetics, and pharmacodynamics were studied. HGF, MET, PD-L1, and ERK expression was evaluated for 9 of 17 patients of the expansion cohort (20 mg/kg). Results: In 39 patients enrolled, no dose-limiting toxicity was observed at 0.3 mg/kg, and the most commonly detected toxicity was generalized edema ( n = 7, 18.9%) followed by pruritis and nausea ( n = 5, 13.5%, each), fatigue, anemia, and decreased appetite ( n = 4, 10.8%, each). No patient discontinued treatment because of adverse events. YYB101 showed dose-proportional pharmacokinetics up to 30 mg/kg. Partial response in 1 (2.5%) and stable disease in 17 (43.5%) were observed. HGF, MET, PD-L1, and ERK proteins were not significant predictors for treatment response. However, serum HGF level was significantly lowered in responders upon drug administration. RNA sequencing revealed a mesenchymal signature in two long-term responders. Conclusion: YYB101 showed favorable safety and efficacy in patients with refractory solid tumors. Based on this phase I trial, a phase II study on the YYB101 + irinotecan combination in refractory metastatic colorectal cancer patients is planned. Conclusion: ClinicalTrials.gov Identifier: NCT02499224

scholarly journals Preparation, Cytotoxicity, and In Vitro Bioimaging of Water Soluble and Highly Fluorescent Palladium Nanoclusters

2020 ◽  
Vol 7 (1) ◽  
pp. 20 ◽  
Author(s):  
Suresh Thangudu ◽  
Poliraju Kalluru ◽  
Raviraj Vankayala
Keyword(s):  
In Vitro Cytotoxicity ◽  
Molecular Probes ◽  
Water Soluble ◽  
Quantum Yields ◽  
Cell Labelling ◽  
Metal Nanoclusters ◽  
Extinction Coefficients ◽  
Cytotoxicity Studies

Fluorescent probes offer great potential to identify and treat surgical tumors by clinicians. To this end, several molecular probes were examined as in vitro and in vivo bioimaging probes. However, due to their ultra-low extinction coefficients as well as photobleaching problems, conventional molecular probes limit its practical utility. To address the above mentioned challenges, metal nanoclusters (MNCs) can serve as an excellent alternative with many unique features such as higher molar extinction coefficients/light absorbing capabilities, good photostability and appreciable fluorescence quantum yields. Herein, we reported a green synthesis of water soluble palladium nanoclusters (Pd NCs) and characterized them by using various spectroscopic and microscopic characterization techniques. These nanoclusters showed excellent photophysical properties with the characteristic emission peak centered at 500 nm under 420 nm photoexcitation wavelength. In vitro cytotoxicity studies in human cervical cancer cells (HeLa) cells reveal that Pd NCs exhibited good biocompatibility with an IC50 value of >100 µg/mL and also showed excellent co-localization and distribution throughout the cytoplasm region with a significant fraction translocating into cell nucleus. We foresee that Pd NCs will carry huge potential to serve as a new generation bioimaging nanoprobe owing to its smaller size, minimal cytotoxicity, nucleus translocation capability and good cell labelling properties.

Phase I Study of IMGN901 in Patients with Relapsed and Relapsed/Refractory CD56-Positive Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3689-3689 ◽  
Author(s):  
Asher Alban Chanan-Khan ◽  
Mecide Gharibo ◽  
Sundar Jagannath ◽  
Nikhil C. Munshi ◽  
Kenneth C Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Phase I ◽  
Phase I Study ◽  
Maximum Tolerated Dose ◽  
Preliminary Evidence ◽  
Clinical Activity ◽  
Weekly Schedule ◽  
Minor Response

Abstract Background: IMGN901 (huN901-DM1) is a novel conjugate of the cytotoxic maytansinoid, DM1, with the humanized CD56-binding monoclonal antibody, N901. Once bound to CD56 on a cancer cell, the conjugate is internalized and releases DM1. About 70% of multiple myeloma (MM) cases have surface expression of CD56. Preclinical investigations demonstrated significant in vitro and in vivo anti-myeloma activity of IMGN901. Objectives: To determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of increasing doses of IMGN901 given for MM on a weekly schedule. Methods: Patients with relapsed or relapsed/refractory MM who have failed at least one prior therapy and have CD56-expressing MM received a single IV infusion of IMGN901 on 2 consecutive weeks every 3 weeks. Patients are enrolled in cohorts of 3 at each dose level, with DLT triggering cohort expansion. Results: Eighteen patients have received IMGN901 to date in this study - 3 patients each at 40, 60, 75, 90, 112, and 140 mg/m2/week. One patient experienced a DLT (grade 3 fatigue) on 140 mg/m2/week, and this cohort is being expanded to enroll up to 6 patients. No patients have experienced serious hypersensitivity reactions or evidence of presence of humoral responses against the huN901 antibody component (HAHA) or against the DM1 component (HADA). Preliminary PK results indicate an approximately linear relationship between dosing and observed maximal serum concentration. A confirmed minor response (MR) was documented in 3 heavily pretreated patients (1 patient each at 60, 90, and 112 mg/m2/week) using the European Bone Marrow Transplant criteria. Durable stable disease was reported at doses of 60, 90, 112, and 140 mg/m2/week. Of the 18 patients treated in the study, eight patients remained on treatment with IMGN901 for at least 15 weeks, five of these 8 patients remained on treatment for at least 24 weeks, and two of these 5 patients remained on treatment for at least 42 weeks. This phase I study provides preliminary evidence of safety as well as clinical activity of IMGN901 in patients with CD56-positive MM who have failed established MM treatments. Updated results of this ongoing study will be presented.

Results of a phase I study to evaluate the feasibility, safety, and biological effects of intravenous administration of wild-type reovirus with docetaxel to patients with advanced malignancies

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e13524-e13524
Author(s):  
S. M. Rudman ◽  
C. Comins ◽  
D. Mukherji ◽  
M. Coffey ◽  
K. Mettinger ◽  
...  
Keyword(s):  
Phase I ◽  
Biological Effects ◽  
Phase 1 ◽  
Maximum Tolerated Dose ◽  
Wild Type ◽  
Systemic Delivery ◽  
Open Label ◽  
Phase Ii Studies

e13524 Background: Reovirus has minimal pathogenicity in humans but selectively replicates in cells with activated Ras. Wild- type reovirus serotype 3 Dearing strain (Reolysin) has selective antitumor activity in vitro, in murine models, and after systemic delivery in humans in phase 1 trials. Synergistic tumour kill has been observed combining reovirus with taxanes in a range of cancer cell lines and in vivo. Methods: Patients were treated in an open-label, dose-escalating, phase I trial and received 3- weekly 75mg/m2 docetaxel i.v. and reovirus i.v. (day 1–5 of first week inclusive). Reovirus was administered at a starting dose of 3x109 tissue culture infectious dose (TCID50) and then increased to 1 x 1010 and 3 x 1010 TCID50. Primary endpoints were to determine the maximum tolerated dose (MTD), dose limiting toxicity (DLT) and to recommend a dose and schedule for future investigation. Secondary endpoints were to evaluate pharmacokinetics, neutralizing antibody development, cell- mediated immune response and anti-tumour activity. Results: 17 patients were treated (15 males, median age 60 years). No MTD has been reached. DLT's observed were G4 neutropenia (and a recurrent perianal abcess) and G3 rise in AST. Other toxicities observed were fatigue, hypotension and neutropenic sepsis. At present, 5 patients remain on treatment. We have observed 2 partial responses (breast and gastric carcinoma) and 10 patients had stable disease as best response. Conclusions: Reovirus is well tolerated when administered in combination with intravenous docetaxel, with predictable toxicity observed. The recommended dose has been defined at 3x1010 TCID50 and phase II studies are planned. Objective radiological evidence of anticancer activity for this combination has been observed. [Table: see text]

Phase I pharmacokinetic (PK) and pharmacodynamic (PD) study of intravenous dimethane sulfonate (DMS612, NSC 281612) in advanced malignancies.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2553-2553
Author(s):  
Susan Elaine Bates ◽  
Sanjeeve Balasubramaniam ◽  
Robert A Parise ◽  
Christina Bryla ◽  
William Bonner ◽  
...  
Keyword(s):  
Dna Damage ◽  
Phase I ◽  
Intravenous Infusion ◽  
Performance Status ◽  
Maximum Tolerated Dose ◽  
Peripheral Blood Lymphocytes ◽  
Ecog Performance Status ◽  
Alkylating Activity

2553 Background: DMS612 is a dimethane sulfonate compound that was identified as preferentially cytotoxic to renal cell carcinoma (RCC) cell lines in a chemical screen of the NCI-60 panel. DMS612 has bifunctional alkylating activity in vitro. Objectives of this first-in-human phase I study included determining the dose-limiting toxicity (DLT), maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), PK and PD of DMS612 administered by 10 minute intravenous infusion on day 1, 8 and 15 of a 28 day cycle. Methods: Eligibility criteria included adults with advanced solid malignancies or lymphoma with ECOG performance status 0-2, life expectancy > 3 months and adequate organ and marrow function. Patients were enrolled using a standard “3+3” dose escalation scheme. Plasma PK of DMS612 and metabolites was assessed by LC-MS/MS. DNA damage PD was assessed by γ-H2AX immunofluorescence. Results: 35 subjects were enrolled (22 male, 13 female) with median age 59 years (41-75). Tumor types included colorectal (8), RCC (4), cervix (2), and urothelial (2). Doses administered were 1.5, 3, 5, 7, 9 and 12 mg/m2. The MTD was determined to be 9 mg/m2, with only one DLT of grade 4 thrombocytopenia in 12 subjects enrolled. The maximum administered dose of 12 mg/m2 was considered to be intolerable after 1 of 3 subjects had grade 4 neutropenia and 1 had prolonged grade 3 thrombocytopenia. Prolonged thrombocytopenia in later cycles was observed in other subjects, including one patient naïve to prior cytotoxic chemotherapy. One subject with RCC had a confirmed partial response at 7 mg/m2. DMS612 was rapidly converted into carboxy, chloroethyl and hydroxyethyl analogues and their glucuronides, some of which retained alkylating activity in vitro. Dose-dependent pharmacodynamic evidence of DNA damage induced by DMS612 in vivo was observed by γ-H2AX immunofluorescance in both peripheral blood lymphocytes and plucked scalp hairs. Conclusions: The MTD of DMS12 administered by intravenous infusion on day 1, 8 and 15 of a 28-day cycle was 9 mg/m2. Pre-clinical and clinical observations suggest that further study of DMS612 in RCC is warranted.

scholarly journals Experimental Validation of a Mathematical Model to Describe the Drug Cytotoxicity of Leukemic Cells

Symmetry ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1760
Author(s):  
Ekaterina Guzev ◽  
Galia Luboshits ◽  
Svetlana Bunimovich-Mendrazitsky ◽  
Michael A. Firer
Keyword(s):  
Mathematical Model ◽  
Drug Efficacy ◽  
In Vitro Cytotoxicity ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Logistic Growth ◽  
Treatment Schedule ◽  
Cytotoxicity Studies

Chlorambucil (Chl), Melphalan (Mel), and Cytarabine (Cyt) are recognized drugs used in the chemotherapy of patients with advanced Chronic Lymphocytic Leukemia (CLL). The optimal treatment schedule and timing of Chl, Mel, and Cyt administration remains unknown and has traditionally been decided empirically and independently of preclinical in vitro efficacy studies. As a first step toward mathematical prediction of in vivo drug efficacy from in vitro cytotoxicity studies, we used murine A20 leukemic cells as a test case of CLL. We first found that logistic growth best described the proliferation of the cells in vitro. Then, we tested in vitro the cytotoxic efficacy of Chl, Mel, and Cyt against A20 cells. On the basis of these experimental data, we found the parameters for cancer cell death rates that were dependent on the concentration of the respective drugs and developed a mathematical model involving nonlinear ordinary differential equations. For the proposed mathematical model, three equilibrium states were analyzed using the general method of Lyapunov, with only one equilibrium being stable. We obtained a very good symmetry between the experimental results and numerical simulations of the model. Our novel model can be used as a general tool to study the cytotoxic activity of various drugs with different doses and modes of action by appropriate adjustment of the values for the selected parameters.

scholarly journals CD72 Nanobody-Based CAR-T Cells Have Potent Anti-Tumor Efficacy in B Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1717-1717
Author(s):  
Matthew A Nix ◽  
William C Temple ◽  
William Karlon ◽  
Donghui Wang ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
In Vitro Cytotoxicity ◽  
B Cell Malignancies ◽  
Cytotoxicity Assays ◽  
Car T Cells ◽  
Car T

Abstract Background: Approximately 50% of pediatric B-ALL patients treated with clinically approved CD19-targeting CAR-T cells do not remain in remission one year after therapy. CD22-targeting CAR-T cells appear to be curative in only a small fraction of CD19-refractory patients and this therapeutic strategy is primarily used as a bridge to stem cell transplant. Additional immunotherapeutic targets thus remain urgently needed. Our laboratory recently used cell surface proteomics to identify CD72 as a B-cell specific marker especially upregulated on poor prognosis, KMT2A/MLL-rearranged B-ALL (Nix et al., Cancer Discovery (2021)). In this published work, we used a best-in-class nanobody library displayed on yeast to develop binders to CD72. We demonstrated for the first time that fully synthetic nanobodies can generate CAR-T cells that are highly potent in vitro and in vivo. While we previously focused on these "nanoCARs" in KMT2A/MLLr B-ALL, in this follow-up study we aimed to 1) further expand our nanoCAR indications to other CD72-expressing B-cell malignancies; 2) biophysically characterize our synthetic nanobodies; 3) evaluate the potential for further humanization of the nanobody binder amino acid sequence while retaining anti-tumor efficacy; and 4) characterize the potency and T-cell immunophenotypes in the context of our lead nanobody binder ("NbD4") placed on different CAR backbones. Methods: Flow cytometry of primary patient samples for CD72 was performed in a CLIA-certified laboratory. NbD4 nanobody was recombinantly expressed in E. coli and biolayer interferometry was used to determine the binding affinity to recombinantly-expressed CD72 extracellular domain. CAR-T cells were generated from peripheral blood donor CD4+ and CD8+ cells (1:1) ratio via lentiviral transduction. In vitro cytotoxicity assays were performed at a range of effector:tumor ratios. In vivo studies were performed in human cell line orthotopic xenografts in NSG mice. 1e6 luciferase-labeled Jeko cells were implanted at Day 0 followed by administration of 4e6 CAR-T cells at Day 6. Tumor burden was assessed by bioluminescence. Results: Flow cytometry on primary non-Hodgkin B-cell lymphoma obtained from fine needle aspiration biopsy (n = 5) confirmed CD72 surface expression (not shown), consistent with RNA-seq across larger cohorts. Biolayer interferometry demonstrated that NbD4 bound with surprisingly low affinity to recombinant CD72 (K D ~800 nM) (Fig. 1A), with both slow on rate (k on 8.38e4 M -1s -1) and rapid off rate (k off 6.82e-2 s -1). This affinity stands in contrast to that reported for FMC63 single chain variable fragment (scFv) used in clinically approved CD19-targeting CAR-T cells (K D 0.3-5 nM), despite similar in vitro and in vivo efficacy of both products. Our NbD4 framework region shows ~82% homology to a human IgG variable heavy domain, significantly higher than FMC63 (~59% homology). We made additional substitutions in the framework domain to increase human homology up to 89%. In vitro cytotoxicity assays with SEM B-ALL cells showed several humanized variants with similar efficacy to NbD4 (Fig. 1B). We further evaluated the impact of placing NbD4 on different CAR backbones, including combinations of CD28 or 4-1BB costimulatory domains and CD8 or IgG4-based transmembrane and hinge regions (Fig. 1C). In vivo, CD72 nanoCARs with Backbone 3 showed significantly increased potency (Fig. 1D). Indeed, tumors treated with Backbone 3 CAR-Ts showed complete tumor clearance and did not develop new tumors even after re-challenge with 1e6 Jeko cells at Day 52 (Fig. 1D). Preliminary characterization of effector and memory CAR-T cell phenotypes before exposure to tumor suggested that Backbone 3 had an increased number of naïve T cells compared to empty CAR and CD19 CAR-T cells (data not shown). Conclusions: Our results demonstrate that our fully synthetic CD72 nanoCARs can effectively eliminate CD72-expressing B-cell malignancy models despite low nanobody binding affinity. Humanized NbD4 variants may serve as clinical candidates with even further reduction in possible immunogenicity of the llama amino acid framework. Alterations to the CAR backbone have a major impact on anti-tumor efficacy and phenotypes of our synthetic nanobodies. CD72-targeting therapies may be effective therapeutics not only KMT2A/MLLr B-ALL but also across a broader spectrum of refractory B-cell malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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