new advances in cellular therapy · purpose. these include ligands, peptides, chimeric ligands,...
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New Advances in Cellular Therapy
Mehrdad Abedi MDAssociate Professor of Medicine
17th Annual Advances in OncologySeptember 30-October 1, 2016
Sacramento, CA
Mehrdad Abedi, M.D.Update on Cellular Therapies for Hematologic Malignancies.
Relevant financial relationships in the past twelve months by presenter or spouse/partner:
Grant/Research Support: CIRM, Kite Pharmaceutical, Celgene, AmgenConsultant/Speaker Programs: Celgene, Millenium, BMS, Gilead
The speaker will directly disclosure the use of products for which are not labeled (e.g., off label use) or if the product is still investigational.
Type of cellular therapy
T cell therapyNK Cell therapy
Stem cell therapyDendritic cell therapy
Mesenchymal Cell Therapy
• Bispecific antibodies
• BsAbs are reagents that combine the specificities of two antibodies in a single molecule.
• Using quadroma technology, two different hybridoma cells are fused to raise a cell that can simultaneously produce the two parental heavy (H) and light (L) chains that join spontaneously by Fc pairing forming
heterodimers• It should also be pointed out that the effector activity of
nonlymphoid cells and natural killer (NK) cells has been exploited in retargeting approaches
At least 40 different ways to generate BsAbs have been described
(A) IgG-based BsAbs
(B) FAb-based BsAbs.
(C) Fv-based BsAbs
(D) scFv-based BsAbs with a protein spacer into the linker
(E) Variants of scFv–Fc
Adaptive Cell Transfer Therapy
Adoptive cell therapy (ACT) is a treatment that uses a cancer patient’s own T lymphocytes with anti-tumour activity, expanded in vitro and reinfused into the patient with cancer.
Adaptive Cell Transfer Therapy
• TIL( Tumor infiltration T-lymphocytes therapy)
• TCR ( T-cell receptor therapy)
• CAR-T (Chimeric antigen receptor T-cell therapy)
Rosenberg, S. A.et al.Use of tumor infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. Preliminary report. N. Engl. J. Med. 319, 1676–1680 (1988).
TIL
3. Infuse the "boosted" T-cells into the patient.
Overview: Adoptive T cell therapy
1. Isolation of TILs or tumor specific T-cells from blood
2. Expand and activate T-cells ex vivo
Target therapy with Tumor specific T cells
Cancer: Melanoma
Autologous tumor infiltrating lymphocytes (TILs); “Live drug”
AdvantagesHigh response rate (>50%),
Long-term remission,
Less toxic & gentler to the patient
Limitation: Extraction of TILs,
Cell manufacturing
Possible alternateT cell Engineering (CAR-T cells)
Rosenberg SA & Dudley ME 2009 Current Opinion of Immunology
Morgan, R. A.et al.Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129 (2006).
TCR
ITAM: immunoreceptor tyrosine-based activation motifTCR complex :TCR, CD3, ζ
TCR
• The Antigen targeting domain• The spacer/hinge domain• The transmembrane domain• The signalling domain
Antigen targeting by CAR molecules most commonly involves the use of scFv. However, several alternative targeting moieties may also serve this purpose. These include ligands, peptides, chimeric ligands, receptor derivatives and single domain antibodies
some reports have suggested that different hinge regions might critically control surface expression levels, construct stability and antigen binding affinity, which directly influence the efficiency of CAR-redirected effector functions
the targeting and signaling properties of CARs, focusing on their effects on T-cell specificity, potency, and safety.
CARs and TCRs have their respective advantages and
disadvantages
• Although the flexibility and “dynamic range” of CARs is attractive, current CARs are limited to recognizing cell surface antigens
• CARs, however, do not require antigen processing and presentation by HLA and are therefore more broadly applicable to HLA-diverse patient populations
• In this regard, CARs provide a broader range of functional effects than transduced T-cell receptors (TCR), wherein strength of signaling, which is for the most part determined by the TCR’s affinity for antigen, is the principal determinant of T-cell fate.
• CARs ﹠TCRs more secure , better targeted , more persistent
3rd generation CARs
Wang et alHum Gen Ther 2007
Zhao et al JI 2009
Wilkie et alJI 2008
Adoptive T cell therapy: CAR-T cells
Antigen specific domain
CAR-T cells (Chimeric antigen receptor-T cells0
T cells transduced with tumor-specific CAR
CAR: Single fusion molecule with antigen specificity plus signaling domain
Three types of CAR: First/second/generations
Based on co-stimulatory receptors
Cancer: Solid tumor & hematological malignancies
Maus M V et al. Blood 2014;123:2625-2635
“Live drug”
Tumor recognition independent of HLA
(no HLA typing needed)
Multiple anti-tumor immuno-modulators can be engineered
Target variety of antigens (protein,
carbohydrate, glycolipid)
Advantages of CAR T cells
What is the ideal TAA?
• Qualities of the “ideal” tumor antigen:
– Expression restricted to the tumor cell population alone
– Restricted expression to tumor and otherwise non-vital tissues
– Expressed by all tumor cells– Expressed on the tumor cell surface– The target antigen is required by the tumor
cell for survival
CAR-targeted TAAs in clinical trials
Jena et al Blood 2010
Clinical significance of CAR-T cells
Target CAR Cancer Objective response
CD19 CAR:CD28-CD3ζ Lymphoma and CLL
N=7: 1CR, 5 PR & 1SD
CAR:CD137-CD3ζ ALL 2CR
CAR:CD28-CD3ζ ALL 5CR
CD20 CAR:CD137-CD28-CD3ζ
NHL N=3: 1PR, 2NED
CEA CAR-CD3ζ (1st gen) Colorectal & breast
N=7: minor responses in two patients
GD2 CAR-CD3ζ (1st gen) Neuroblastoma N=19: 3CR
ERBB2 CAR:CD28-CD137-CD3ζ
Colorectalcancer
N=1, patient died
Kershaw et. al. 2013 Nature Reviews cancer
Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. Aug 25 2011;365:725-33
Expression of CD19 and other B cell markers on B lineage
cellspreB-ALL
B cell lymphomas and leukemias myelomas
Stem Cell pre B immature B mature B plasma cellpro B
CD19
CD22
CD20
Figure 1 Clinical Response in the Patient.
Figure 1 Clinical Response in the Patient.
Before treatment 6 months after treatment
Response in Patient with Refractory DLBCL
Depth of Best Response in NCI B Cell Lymphoma Study
6confidential
Figure 2 Serum and Bone Marrow Cytokines before and after Chimeric Antigen Receptor T-Cell Infusion.
Figure 3.Expansion and Persistence of Chimeric Antigen Receptor T Cells In Vivo.
Failure• Morgan RA, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR
gene therapy. J Immunother 2013;36:133–151.
• Morgan RA, Yang JC, Kitano M, Dudley ME,Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol Ther 2010;18:843–851.
• Parkhurst MR, et al. T cells targeting carcinoembryonic antigen can mediateregression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011;19:620–626.
• Brentjens R, Yeh R, Bernal Y, Riviere I, SadelainM. Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial. Mol Ther 2010;18:666–668.
Challenges of CAR-T
Target selection
Optimize costimulatory signaling of T cell effector functions
Toxicities (on-target but off-tumor toxicity)(The on-target toxicities result from the inability of engineered T cells to distinguish between normal cells and cancer cells that express the targeted Ag.)
Cytokine release syndrome
Tumor lysis syndrome
Neurologic toxicities
Toxicities
Toxicities
On target/off tumor toxicities
Metastatic colon cancer patient died after 5 days of infusion of ERBB2+CAR-T cells
Low levels of ERBB2 express on lung epithelium (lung tox)
Renal cell carcinoma: 5/11 patients developed liver toxicity
Cytokine syndrome
Elevated levels of pro-inflammatory cytokines
Treatable by anti-IL-6mAb and steroids
Challenges of CAR-T cells
Efficacy & Long-term persistenceSubtypes of CD4+T cells (Th1, Th2, Th17, Th9 cells),
CD8+T cells
naïve, central memory; long-term
effector; active but short lived
Patient conditioning before ACTReduced-intensity or non-myeloablative
Increased intensity myelo ablative
Tumor targetTarget antigen is critical determinant for efficacy & safety
Ideal target uniquely express on tumor cells or on cells which are not essential for survival
Trafficking of CAR T cells to tumorExpression of addressins
Route of CAR-T cell infusion
Intra-tumoral/intravenous
Optimal co-stimulation of T cells
Determinants of successful ACT: CAR-T cells
CART Programs at Academic Centers
Kenderian et al. BBMT in press.
Moving Forward with CAR-T Cells
Development of CAR modified T cellcancer therapy
• CAR design• CAR modified T cell trafficking• CAR modified T cell persistence• CAR modified T cell perseverance• Conditioning regimen including
Lymphodepletion
Moving Forward: Armored CARs
Moving Forward
New CAR T cell Strategies• Combination with immune
stimulator drugs• Different CAR T cell populations
• Cytotoxic T-cell (CD8)
• Helper T-cell (CD4)
• Regulatory/suppressor T-cell
• Memory T-cell
Classfication of T-cell
IL23R
Adoptive T cell therapy: Right T cell population?
CD4+ T cells
Engineering Th9 cells: TAA specific Tumor model
OT2 TCR transgenic mice (CD4 cells recognize ova)
B16F10-Ova cells Follow tumor development
Normal WT or Rag1-/-
CD4+CD25-CD62L+
Th17
Th9TGFβ + IL-4
TGFβ + IL-6
Naïve OT2
Engineer tumor specific Th9 cells
Generation of Ovalbumin expressing B16 tumor cells(Lentiviral method)
Phase I Trial of Stem Cell Gene Therapy for HIV in AIDS Lymphoma Patients
Why Is It So Difficult to Cure HIV?
Microglial and Kupffer cells,
http://abcnews.go.com/Health/french-hiv-study-means-hiv-babys-cure/story?id=18741318#.UZFht7VOQrU
http://www.cnn.com/2013/03/18/health/hiv-functional-cure
Candidate Product
an shRNA against CCR5, Cell entry phase
a chimeric Trim5α: pre-integration stage by disrupting viral capsid uncoating
a TAR decoy: post-integration/pre-transcription
All transferred by a single self-inactivating lentiviral vector
Gene modified HSC with a triple combination of anti-HIV molecules:
Down regulation of CCR5 expression on 1TAX cells
pre-selective anti-HIV vectors
CD34+ HSCs
vector transduced HSCs
purification
purified HIV-resistant HSCs
transduction
Pre-selective Approach
Barclay et al. 2014 Stem Cells
Enrichment of 1TAX vector transduced cells
Phenotypic profile of 1TAX vector transduced peripheral blood T cells
HIV-1 challenge of 1TAX HSPC derived macrophages
Maintenance of human CD4+ T cells in vivo in 1TAX cell engrafted NRG mice infected with HIV-1
Study Identifier or Title
Study Objective(s) Test Article and Manufacturing Process Used
Animal Model and Number of Animals pergroup
Key Outcome(s)
Immunogenicity of chimeric T5A To determine any immunogenicity to chimeric T5A Research grade NA No reaction detected (t-test, p>0.05) In vitro toxicity To determine any toxicity in transduced PBMCs Research grade NA No toxicity detectedIn vitro CFU assay To determine any toxicity in transduced CD34+ HPCs Clinically equivalent NA No toxicity detected (t-test, p>0.05)Purification of transduced cells To determine the levels of enrichment of transduced HPC Clinically equivalent NA Purification of >95% of transduced cellsIn vitro CFU/IL2 assay To determine if the addition of IL2 increased transduced cell
proliferationClinically equivalent NA No increased proliferation (t-test, p>0.05)
Proto-oncogene QPCR To determine any upregulation of proto-oncogenes Research grade NA No increased expression (t-test, p>0.05)CD122/CD132 flow cytometry To determine any upregulation of CD122 or CD132 Clinically equivalent NA No upregulation of CD122 of CD132 (t-
test, p>0.05)In vitro immortalization To determine if immortalization occurs with transduced cells Clinically equivalent NA No immortalization had occurred
Phenotypic analysis of HPC derived macrophages To determine if transduced cells have a normal macrophage phenotype Clinically equivalent NA Transduced macrophages had a normal macrophage phenotype (t-test, p>0.05)
Anti-HIV gene expression To determine the expression of anti-HIV genes Clinically equivalent NA Detection of expression of all three genes
Stability of vector To determine the stability of the vector in transduced cells Clinically equivalent NA No deletions or rearrangements were detected
CCR5 downregulation To determine the levels of CCR5 downregulation Clinically equivalent NA >93% downregulation of CCR5 (t-test, p<0.05)
In vivo engraftment and multi-lineage hematopoiesis
To determine the in vivo engraftment and multi-lineage hematopoiesis levels of transduced cells
Clinically equivalent NRG (N=10) Normal engraftment levels were observed in the peripheral blood + lymphoid organs (t-test, p>0.05)
Tumorigenicity To determine the tumorigenicity of transduced cells Clinically equivalent NRG (N=8) No tumors were detectedCytokine secretion profile To determine if engrafted human T cells are functionally normal Research grade NRG (N=3) Normal cytokine profiles (t-test, p>0.05)
Karyotyping To determine if transduced cells have a normal karyotype Research grade NA A normal karyotype was observedSequencing To determine if the sequence of integrated vector is correct Clinically equivalent NA Correct sequence was observedSafety of rapamycin To determine the safety of rapamycin treated CD34+ HSC Clinically equivalent NA No toxicity observed (t-test, p>0.05)In vitro/ex vivo HIV-1 challenge To determine the levels of protection of transduced cells Clinically equivalent NA >3 logs inhibition of HIV-1 infection (t-
test, p<0.05)In vivo HIV-1 challenge To determine if transduced and engrafted human immune cells
inhibited HIV-1Clinically equivalent NRG (N=14) lower levels of HIV-1 viremia (t-test,
p<0.05)Escape mutants To determine if any viral escape mutants arose in HIV-1 challenge
experimentsResearch grade NA No escape mutants were detected
Selective survival To determine a selective survival advantage of transduced cells Clinically equivalent NRG (N=8) A selective survival advantage was detected with transduced cells (t-test, p<0.05)
HIV-1 integration To determine if HIV-1 challenge virus was able to create provirus Research grade NA No integration of HIV-1 was detected
CD4+ cell maintenance To determine if normal CD4+ cell levels could be maintained in transduced cell engrafted mice
Clinically equivalent NRG (N=14) Normal CD4+ cell levels were observed in mice engrafted with transduced cells (t-test, p<0.05)
Lentiviral transduction % To determine if rapamycin treatment increased vector transduction Clinically equivalent NA A 3-4 fold increase in transduction (t-test, p<0.05)
A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-selected CD34+ Cells
NCI Protocol: AMC-097
Combinatorial anti-HIV lentivirus:• an shRNA against CCR5• a chimeric Trim5α• a TAR decoy• CD25 preselection of
Phase I Study of Anti-HIV Gene Modified Hematopoietic Stem cells Transplant in Patient With HIV Related
Lymphomas to Cure Both Their Lymphoma and HIV
Regulatory:NIH RAC and FDA IND approval have been obtainedFunding:NIH CTEP and CIRM
High-grade non-Hodgkin’s lymphoma, meeting one of the following criteria
• In partial remission,• Relapsed after initial complete remission,• Failed induction therapy, but responds to salvage therapy (i.e., chemosensitive disease),• In complete remission with high-risk features as specified by the International Prognostic Index.
Advanced stage follicular lymphoma, that have failed at least two lines of therapy multi-agent chemotherapy, but responds to salvage therapy (i.e., chemosensitive disease)
Advanced stage Mantle cell lymphoma with Ki-67 > 10% in first complete remission
Hodgkin’s lymphoma, meeting one of the following criteria
• In first, or greater relapse after initial complete remission,• In partial remission,• Failed induction therapy, but responds to salvage therapy (i.e., chemosensitive disease).
Burkitt’s lymphoma, meeting one of the following criteria:•In second complete remission after relapse following initial complete remission,•Failed induction therapy, but responds (very good partial remission, complete remission, or near complete remission) to salvage therapy (i.e., chemosensitive disease).
Plasmoblastic lymphomas, or peripheral T cell lymphoma (with the exception of ALK+ type in first or second complete remission) (timeline 4 months prior to start of trial).
The primary endpoint of the study is:
Safety, defined as timely engraftment (the collective establishment of a persistent absolute neutrophil count of at least 500 cells/mm3 and platelet count of 20,000 cells/mm3 without transfusion for 3 consecutive days) at one month post transplant, in the absence of any study candidate specific grade 3 and 4 non-hematopoietic organ toxicity or any clonal expansion;
Major Secondary Objectives
• Efficacy of the candidate product, defined as establishment of > 5% mononuclear blood cells expressing anti-HIV genes in the peripheral blood at 3 months post-transplant.
• To determine the presence, quantity, and duration of gene modified HIV-1 resistant peripheral blood cells and gut mucosal immune cells.
• To study the integration sites of vector sequences in circulating cells.• Minor Secondary Objectives• To study progression-free survival.• To study overall survival.• To study complete response rate and duration.• To study partial response rate and duration.• To study time to neutrophil engraftment (first of 3 consecutive days of ANC > 500
cells/mm3.• To study time to platelet engraftment (first of 3 consecutive days of platelets > 20,000
cells/mm3 without platelet transfusions 7 days prior).• To study hematologic function at Day 100 (ANC > 1500, Hb > 10g/dl without transfusion
and platelets > 100,000)• To study CD4 recovery at the conclusion of the trial.• To study safety in terms of toxicities, infections, transfusions, and infusion-related
reactions.• To study HIV-1 viral load over time.• To study persistence of vector-transduced cells over time.
Study Cohorts Ratio of Transduced versus Untransduced Stem Cells
1 1:1
2 5:1
3 1:0
Study Cohorts
Study Cohorts
Ratio of Transduced vs Un-transduced Stem
Cells
Minimum Number of Transduced/Un-transduced
Cells Acceptable. CD34 cells/kg
Goal for Number of Transduced Cells.
CD34 cells/kg
1 1:1 1 X 106:1 X 106 2-5 X 106: 2-5 X 106
2 5:1 2 X 106:1 X 106 5 X 106:1 X 106
3 1:0 2 X 106: 0 5-10 X 106: 0
How Many CD34 Do We Need?
• At or after six months post transplant• Voluntarily • Eight-week period• Only in the third cohort • CD4 count of 500 or higher with no detectable viral load by single copy PCR.
ART withdrawal
Immunology Study in HIV-Stem Cell Protocol
I. Reconstitution of Immune System in Peripheral Blood:1. Subsets and Differentiation of CD4 and CD8 T Lymphocytes:
Developed from our previous and current studies, flow cytometric assays will be used:
a. Determine the memory and naïve CD4 and CD8 T cells as well as the T cell activation[CD3, CD4, CD8, CCR7, CD45RA, CD38, HLA-DR, CD69, CD71 and viable cell stain]
b. Determine the Treg, NK and NK-T cell subsets:[CD3, CD4, CD8, CD25, IL-7R, FoxP3, CD16, CD56 and viable cell stain]
*expression of homing molecules CD18, CD31, CD54 may be added to this test.
These assays will be performed after stem-cell transplant to monitor the re-establishment of T cell system.
2. Functional Responses Upon Reconstitution of T Cell ImmunityThe following studies are proposed, primarily to measure the cytokine responses:a. Total Th1/Th2/Th17 subsets will be measured after mitogen stimulation (1-2 months after transplant)
[CD3, CD4, CD8, and intra-cellular IFNg, TNFa, IL2, IL4, IL10]
b. Antigen specific T cell responses will be measured after stimulations with CEF and HIV-gag peptides[proposed flow cytometric assay: CD3, CD4, CD8, and intra-cellular IFNg, TNFa, IL2][alternative assay, a dual-color ELISpot Assay to evaluate IFNg/IL2 producing T cells, if PBMC cells are limited]
c. If adequate PBMC are collected, proliferation responses will be tested using CFSE assays
Immunology Study in HIV-Stem Cell Protocol
II. Reconstitution of T Cell Immune System in Gastrointestinal-associated lymphoid tissue:
1. After stem cell transplant (2-4 months?), GI mucosal biopsy samples will be obtained.2. Biopsy tissues digested with Collagenese will be used for phenotyping
and functional assay:a. Determine the memory and naïve CD4 and CD8 T cells as well as the
T cell activation[CD3, CD4, CD8, CCR7, CD45RA, CD38, HLA-DR, CD69, CD71 and
viable cell stain]b. Ag-specific T cell responses will be measured after mitogen, CEF
and HIV stimulations[CD3, CD4, CD8, and intra-cellular IFNg, TNFa, IL2, IL4, IL10]
[alternatively, ELISpot will be used to evaluate IFNg producing T cells]
AIDS Malignancy Consortium
Sponsored by NCI 25 US Sites 9 International Sites
initial CD34 %
total MNCsb
initial CD34+
isolated CD34+
transduced CD34+
CD34+post-transduction
CD34+post CD25 selection
% yield from initial CD34+
sample
chemoExpt 1 1.16 1.12x10^8 1.3x10^6 1.48x10^6 1.44x10^6 1.6x10^6 1.08x10^6 83.1 mobilized/lymphoma
G+Plerixafor/multipleExpt 2 0.35 4.3x10^8 1.5x10^6 1.1x10^6 1.1x10^6 1.07*10^6 1.84x10^6 122.7 myeloma
chemoExpt 3 1.11 8.32x10^7 923,520 1.02x10^6 1.5x10^6 1.16x10^6 620,000 67.1 mobilized/lymphoma
G+Plerixafor/multipleExpt 4 0.37 4.44x10^8 1.64x10^6 1.45x10^6 1.5x10^6 1.26x10^6 1.15x10^6 70.1 myeloma
Expt 5 4.8 5.12x10^7 2.46x10^6 2.13x10^6 2.35x10^6 1.875x10^6 1.3x10^6 52.8 cord blood
G+Plerixafor/multipleExpt 6 0.23 5.79x10^8 1.33x10^6 870,000 1.57x10^6 1.27x10^6 552,056 41.5 myeloma
average%CD34
recoveredc89.65
Experiments MNCreceived
Total number of CD34 cells received
Total number of CD34scollected from the CD34 column (percentage of cell recovery)
Total number of CD34s collected after CD25 column (percentage of cell recovery)
Purity for the CD25+cells
1 4.7x10^10 3.57x10^8 1.55x10^8 (43.4%) 3.3x10^6 (19%) 25.4%
2 (label check expt) 5.8x10^10 5.1x10^8 4.28x10^8 (83.9%) N/A N/A
3 5.8x10^10 1.18x10^9 9.20x10^8 (78.0%) 6.3x10^7 (16.7%) 97.4%
4 5.7x10^10 2.10x10^8 1.49x10^8 (71.0%) 3.65x10^7 (79.9%) 86.7%
CTS1-08289Patient #1
summary of cell processing andrelease tests
MB is a 50 years old WM with relapsed Hodgkin's lymphomaPatient had a very poor collection with G+Mozobil.Febrile syndrome after collection.Disease relapsed while we were waiting for the second round of collectionWent to remission with ARA-C+etoposidePoor collection after chemomobilization.Added mozobil and given bid G-CSF with improved collectionWe initially had a problem with high granulocyte content but troubleshooted around this problem. CD34+ recovery, purity and viability increased.Transductions were high enough, and end CD34+/CD25+ cell purity was good. However, CD34+/CD25+ recovery was again low.
Patient #1 Mobilized Leukopaks1932-1936 are patient leukopak ID#s obtained on consecutive days. Due to poor CD34+ purity,viability, and recovery, we did not use leukopaks 1932-1934 for further transduction andseparation.
Leukopak id# Initial
CD34%
Theoretical yield
Granulocyte %
Recovery
% CD34 purity
% recovered
% viable
changed protocol to include increased IVIG with apreincubation period prior to labeling
1932/1933 1934 1935 1936
0.21 0.28 0.30 0.41
2.9x10^8 2.5x10^8 3.7x10^8 5.81x10^8
42 53 84 46
4.9x10^7 1.27x10^8 2.9x10^8 2.11x10^8
65.74 48.0 82.2 90.5
11.2 24.3 64.4 32.9
81.79 69.82 96.6 98.5
1935:1936:
cells transduced = 2.6x10^8 cells transduced = 1.8x10^8
Transduction efficiency:1935 = 23.6%1936 = 31.2%
Total CD34+/CD25+ cells:1935 = 6.136x10^71936 = 5.616x10^7
Theoretical yield at 100% efficiency= 1.18x10^8CD34+/CD25+ cellsOnly need to recover 7.0x10^7 (59.3%) for the 99kg patient
Recovered ~2.5x10^7 CD34+/CD25+ cells
• Only used the cells from leukopaks 1935 and 1936 dueto bad
purity and viability from others
How these genetic modification will affect hematopoietic cells
Immunological Reaction: elimination Differentiation issues Chemokine-cytokine-integrin disturbances
resulting in motility/homing/functional issues
ART Immunological Non-respondersFuture Directions
Very low doses of radiation can results in high level of engraftment
Principal InvestigatorAbedi
External Advisory BoardKohn, Rossi, Torbett,
Volberding
DNA monitoring
CoreAnderson
Regulatory LeadBauer
CMC (Bauer)• GMP facilityoVector productionoProgenitor LaboQA/ Control
CMC/Product Development Lead
Richman
Immunology Specialty
CorePollard
Clinical Project Manager TBD
Clinical LeadAbedi
HIV teamUC Davis
UCSF
Transplant team
UC DavisUCSF
FDA
Preclinical team Clinical team
Community AdvocateTBD
Recruitment
Co-PIAnderson
IRB
Preclinical Project Manager Bauer
Clinical Operations• CRC UCD (Quirch, Eddings)• CRC UCSF (Boyer, Murray,
Romano)• Data management (TBD)
Clinical Operations Core• CTSU• DSMB (TBD)• Biostatistics (Beckett)
Regulatory• FDA• NIH RAC• UC Davis Biosafety
Biologics Consulting Group, Inc
Miller
Acknowledgement